Gene families associated with stomach cancer

ABSTRACT

The invention relates generally to the changes in gene expression in stomach cancer. The invention relates specifically to human gene families that correspond to mRNA species that are differentially expressed in cancerous stomach tissue compared to normal stomach tissue.

TECHNICAL FIELD

The invention relates generally to the changes in gene expression instomach tissue from stomach cancer patients compared to normal stomachtissue. The invention specifically relates to human gene families whichare differentially expressed in advanced gastric cancers and othermalignant neoplasms compared to normal tissue.

BACKGROUND ART

Stomach Cancer

In the United States, approximately 24,000 new cases of stomach cancer,or gastric cancer, are diagnosed every year. Although the incidence ofstomach cancer has declined significantly in the last 60 years, it isstill a serious disease caused by factors that remain elusive. Undersimilar circumstances, some people develop stomach cancer and others donot.

Stomach cancer usually occurs in people over the age of 55 and is twiceas common in men as in women. This type of cancer is not prevalent inthe United States, but it is much more prevalent in Japan, Korea, LatinAmerica and parts of Eastern Europe, where people eat more foods thatare preserved by drying, pickling, smoking or salting. Conversely,consuming fresh fruits and vegetables may protect against this disease.

Stomach cancer can develop in any part of the stomach and spreadthroughout the stomach and/or to other organs. The cancer may also growalong the stomach wall and spread to the esophagus or small intestine.If the cancer grows through the stomach wall, it can extend to nearbylymph nodes, the liver and the pancreas and the colon. Stomach cancercan spread even farther, to the ovaries, lungs and distant lymph nodes.When stomach cancer metastasizes to another part of the body, thesetumor cells are of the same type as those in the original tumor. Inother words, metastasized cells in the liver are still stomach tumorcells. Such tumor cells that spread to an ovary, establishing one ormore ovarian tumors, are known as Krukenberg tumors and are composed oftransformed stomach cells, not ovarian cells.

Because the symptoms of stomach cancer are non-specific, this cancer isdifficult to detect in its early stages. Symptoms include indigestion,heartburn, abdominal pain, nausea and vomiting, diarrhea orconstipation, loss of appetite, weakness and fatigue, and bleeding whichis detected by blood in the stool or by the affected person vomitingblood. Diagnosis is usually performed by x-rays of the uppergastrointestinal tract and esophagus, the x-rays taken after the patienthas consumed a liquid barium tracer. Endoscopy of the stomach andesophagus, with a gastroscope, can also be performed. If abnormal tissueis found, it can be biopsied through the gastroscope. Should the biopsyspecimen show cancerous cells, surrounding lymph nodes are thenbiopsied, and surrounding organs, such as the liver and pancreas, areexamined via CT scan to determine the extent or stage of the disease.Treatment methods for stomach cancer are similar to those employed inother types of cancer-removal of the affected organ partial or totalgastrectomy), possibly with removal of nearby lymph nodes as well,chemotherapy, radiation therapy and immunotherapy (stimulating immunesystem components that attack cancer cells)(http://cancernet.nci.nih.gov/cancertypes.html). As early stomach cancercauses few symptoms, diagnosis is not usually made before the advancedstages of the disease, where treatments are less effective.

Molecular Changes in Stomach Cancer

Little is known about the molecular changes in stomach cells associatedwith the development and progression of stomach cancer. Accordingly,there exists a need for the investigation of the changes in geneexpression levels, as well as the need for the identification of newmolecular markers associated with the development and progression ofstomach cancer. Furthermore, if intervention is expected to besuccessful in halting or slowing the progression of stomach cancer,means of accurately assessing the early manifestations of this diseaseneed to be established. One way to accurately assess the earlymanifestations of stomach cancer is to identify markers which areuniquely associated with disease progression (see for example Kim et al.(2001), Oncogene 20: 4568-4575). Likewise, the development oftherapeutics to prevent or stop the progression of stomach cancer relieson the identification of genes responsible for cancerous transformationand growth in the stomach.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of new gene familiesthat are differentially expressed in advanced gastric cancer (AGC) andother malignant neoplasms compared to normal tissue. The inventionincludes an isolated nucleic acid molecule comprising SEQ ID NO: 3, 5,7, 9, 11, 13, 17 or 19; an isolated nucleic acid molecule that encodesthe amino acid sequence of SEQ ID NO: 4, 14 or 18; an isolated nucleicacid molecule that encodes a protein that is expressed in stomach cancerand that exhibits at least about 92% nucleotide sequence identity overthe entire length of SEQ ID NO: 3 or 17, an isolated nucleic acidmolecule that encodes a protein that is expressed in stomach cancer andthat exhibits at least about 95% nucleotide sequence identity over theentire length of SEQ ID NO: 13, and an isolated nucleic acid moleculecomprising the complement of any of the aforementioned nucleic acidmolecules.

The present invention further includes the nucleic acid moleculesoperably linked to one or more expression control elements, includingvectors comprising the isolated nucleic acid molecules. The inventionfurther includes host cells transformed to contain the nucleic acidmolecules of the invention and methods for producing a proteincomprising the step of culturing a host cell transformed with a nucleicacid molecule of the invention under conditions in which the protein isexpressed.

The invention further provides an isolated polypeptide selected from thegroup consisting of an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 18, an isolatedpolypeptide comprising a fragment of at least 10 amino acids of SEQ IDNO: 6, 8, 10 or 12, an isolated polypeptide comprising conservativeamino acid substitutions of SEQ ID NO: 6, 8, 10 or 12 and an isolatedpolypeptide comprising naturally occurring amino acid sequence variantsof SEQ ID NO: 6, 8, 10 or 12. Polypeptides of the invention also includepolypeptides with an amino acid sequence having at least about 90% aminoacid sequence identity with the sequence set forth in SEQ ID NO: 4,preferably at least about 92-95%, and more preferably at least about95-98% sequence identity with the sequence set forth in SEQ ID NO: 4.Polypeptides of the invention also include polypeptides with an aminoacid sequence having at least about 50%, 60%, 70% or 75% amino acidsequence identity with the sequence set forth in SEQ ID NO: 6, 8, 10 or12, preferably at least about 80%, more preferably at least about90-95%, and most preferably at least about 95-98% sequence identity withthe sequence set forth in SEQ ID NO: 6, 8, 10 or 12. Polypeptides of theinvention also include polypeptides with an amino acid sequence havingat least about 95% and at least about 92% amino acid sequence identitywith the sequence set forth in SEQ ID NO: 14 and SEQ ID NO: 18,respectively.

The invention further provides an isolated antibody or antigen-bindingantibody fragment that specifically binds to a polypeptide of theinvention, including monoclonal and polyclonal antibodies.

The invention further provides methods of identifying an agent whichmodulates the expression of a nucleic acid molecule encoding a proteinof the invention, comprising: exposing cells which express the nucleicacid molecule to the agent; and determining whether the agent modulatesexpression of said nucleic acid molecule, thereby identifying an agentwhich modulates the expression of a nucleic acid molecule encoding theprotein.

The invention further provides methods of identifying an agent whichmodulates the level of or at least one activity of a protein of theinvention, comprising: exposing cells which express the protein to theagent; and determining whether the agent modulates the level of or atleast one activity of said protein, thereby identifying an agent whichmodulates the level of or at least one activity of the protein.

The invention further provides methods of identifying binding partnersfor a protein of the invention, comprising the steps of exposing saidprotein to a potential binding partner; and determining if the potentialbinding partner binds to said protein, thereby identifying bindingpartners for the protein.

The present invention further provides methods of modulating theexpression of a nucleic acid molecule encoding a protein of theinvention, comprising the step of administering an effective amount ofan agent which modulates the expression of a nucleic acid moleculeencoding the protein. The invention also provides methods of modulatingat least one activity of a protein of the invention, comprising the stepof administering an effective amount of an agent which modulates atleast one activity of the protein of the invention.

The present invention further includes non-human transgenic animalsmodified to contain the nucleic acid molecules of the invention, ornon-human transgenic animals modified to contain the mutated nucleicacid molecules such that expression of the encoded polypeptides of theinvention is prevented.

The present invention also includes non-human transgenic animals inwhich all or a portion of a gene comprising all or a portion of SEQ IDNO: 3, 5, 7, 9, 11, 13 or 17 has been knocked out or deleted from thegenome of the animal.

The invention further provides methods of diagnosing stomach cancer orother malignant neoplasms, comprising the steps of acquiring a tissue,blood, urine or other sample from a subject and determining the level ofexpression of a nucleic acid molecule of the invention or polypeptide ofthe invention.

The invention further includes compositions comprising a diluent and apolypeptide or protein selected from the group consisting of an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6, 8,10, 12, 14 or 18, an isolated polypeptide with an amino acid sequencehaving at least about 90% amino acid sequence identity with the sequenceset forth in SEQ ID NO: 4, preferably at least about 92-95%, and morepreferably at least about 95-98% sequence identity with the sequence setforth in SEQ ID NO: 4, an isolated polypeptide comprising a fragment ofat least 10 amino acids of SEQ ID NO: 6, 8, 10 or 12, an isolatedpolypeptide comprising conservative amino acid substitutions of SEQ IDNO: 6, 8, 10 or 12, naturally occurring amino acid sequence variants ofSEQ ID NO: 6, 8, 10 or 12, ah isolated polypeptide with an amino acidsequence having at least about 50%, 60%, 70% or 75% amino acid sequenceidentity with the sequence set forth in SEQ ID NO: 6, 8, 10 or 12,preferably at least about 80%, more preferably at least about 90-95%,and most preferably at least about 95-98% sequence identity with thesequence set forth in SEQ ID NO: 6, 8, 10 or 12, an isolated polypeptidewith at least about 95% amino acid sequence identity with the sequenceset forth in SEQ ID NO: 14, or an isolated polypeptide with at leastabout 92% amino acid sequence identity with the sequence set forth inSEQ ID NO: 18.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 FIG. 1 is a diagram showing the sequence differences between SEQID NO: 1 (clone AD12) and SEQ ID NO: 3 (clone CH4), which are splicevariants of the gene designated LBFL301.

FIG. 2 FIG. 2 is a hydrophobicity plot of the protein encoded by theopen reading frame of LBFL301, variant AD12 (SEQ ID NO: 2). Analysis wasperformed according to the methods of Kyte-Doolittle and Goldman et al.

FIG. 3 FIG. 3 is a hydrophobicity plot of the protein encoded by theopen reading frame of LBFL301, variant CH4 (SEQ ID NO: 4). Analysis wasperformed according to the methods of Kyte-Doolittle and Goldman et al.

FIG. 4 FIG. 4 is a hydrophobicity plot of the protein encoded by thelongest of the open reading frames of LBFL304 (SEQ ID NO: 6). Analysiswas performed according to the methods of Kyte-Doolittle and Goldman etal.

FIG. 5 FIG. 5 is a hydrophobicity plot of the protein encoded by theopen reading frame of LBFL305 (SEQ ID NO: 14). Analysis was performedaccording to the methods of Kyte-Doolittle and Goldman et al.

FIG. 6 FIG. 6 shows the relative alignment positions of the threeLBFL306 clones.

FIG. 7 FIG. 7 is a hydrophobicity plot of the protein encoded by theopen reading frame of clone no. LBFL306-EF3 (SEQ ID NO: 18). Analysiswas performed according to the methods of Kyte-Doolittle and Goldman etal.

FIG. 8 FIG. 8 is a hydrophobicity plot of the protein encoded by theopen reading frame of clone no. LBFL306-GC7 (SEQ ID NO: 20). Analysiswas performed according to the methods of Kyte-Doolittle and Goldman etal.

FIG. 9 FIG. 9 is a hydrophobicity plot of the protein encoded by theopen reading frame of clone no. LBFL306-GE2 (SEQ ID NO: 22). Analysiswas performed according to the methods of Kyte-Doolittle and Goldman etal.

BEST MODE FOR CARRYING OUT THE INVENTION

I. General Description

The present invention is based in part on the identification of new genefamilies that are differentially expressed in cancerous human stomachtissue and other malignant neoplasms compared to normal human tissue.These gene families include the human cDNA of SEQ ID NOS: 1, 3, 5, 7, 9,11, 13, 17, 19 and 21.

The genes and proteins of the invention may be used as diagnostic agentsor markers to detect stomach cancer or to monitor the progression ofstomach cancer in a sample. They can also serve as a target for agentsthat modulate gene expression or activity. For example, agents may beidentified that modulate biological processes associated with tumorgrowth, including the hyperplastic process of stomach cancer.

II. Specific Embodiments

A. The Proteins Associated with Stomach Cancer

The present invention provides isolated proteins, allelic variants ofthe proteins, and conservative amino acid substitutions of the proteins.As used herein, the “protein” or “polypeptide” refers, in part, to aprotein that has the human amino acid sequence depicted in SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or 18. The terms also refer to naturally occurringallelic variants and proteins that have a slightly different amino acidsequence than that specifically recited above. Allelic variants, thoughpossessing a slightly different amino acid sequence than those recitedabove, will still have the same or similar biological functionsassociated with these proteins.

As used herein, the families of proteins related to the human amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18 include proteinsthat have been isolated from organisms in addition to humans. Themethods used to identify and isolate other members of the family ofproteins related to these proteins are described below.

The proteins of the present invention are preferably in isolated form.As used herein, a protein is said to be isolated when physical,mechanical or chemical methods are employed to remove the protein fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated protein.

The proteins of the present invention further include splice variantsand insertion, deletion or conservative amino acid substitution variantsof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18. As used herein, aconservative variant refers to alterations in the amino acid sequencethat do not adversely affect the biological functions of the protein. Asubstitution, insertion or deletion is said to adversely affect theprotein when the altered sequence prevents or disrupts a biologicalfunction associated with the protein. For example, the overall charge,structure or hydrophobic/hydrophilic properties of the protein, incertain instances, may be altered without adversely affecting abiological activity. Accordingly, the amino acid sequence can bealtered, for example to render the peptide more hydrophobic orhydrophilic, without adversely affecting the biological activities ofthe protein.

Ordinarily, the allelic variants, the conservative substitutionvariants, and the members of the protein family encoded by LBFL301, willhave an amino acid sequence having at least about 50%, 60%, 70% or 75%amino acid sequence identity with the sequence set forth in SEQ ID NO: 2or 4, more preferably at least about 80-90%, even more preferably atleast about 92-95%, and most preferably at least about 95-98% sequenceidentity. The allelic variants, the conservative substitution variants,and the members of the protein family encoded by LBFL304, will have anamino acid sequence having at least about 50%, 60%, 70% or 75% aminoacid sequence identity with the sequence set forth in SEQ ID NO: 6, 8,10 or 12, more preferably at least about 80%, even more preferably atleast about 90-95%, and most preferably at least about 99 or 99.5%sequence identity. The allelic variants, the conservative substitutionvariants, and the members of the protein family encoded by LBFL305 orLBFL306, will have an amino acid sequence having at least about 50%,60%, 70% or 75% amino acid sequence identity with the sequence set forthin SEQ ID NO: 14 or 18, more preferably at least about 80-90%, even morepreferably at least about 92-94%, and most preferably at least about95%, 98% or 99% sequence identity. Identity or homology with respect tosuch sequences is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or 18 after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent homology, and notconsidering any conservative substitutions as part of the sequenceidentity (see section B for the relevant parameters). Fusion proteins,or N-terminal, C-terminal or internal extensions, deletions, orinsertions into the peptide sequence shall not be construed as affectinghomology.

Thus, the proteins of the present invention include molecules having theamino acid sequence disclosed in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or18; fragments thereof having a consecutive sequence of at least about 3,4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of theseproteins; amino acid sequence variants wherein one or more amino acidresidues has been inserted N- or C-terminal to, or within, the disclosedcoding sequence; and amino acid sequence variants of the disclosedsequence, or their fragments as defined above, that have beensubstituted by at least one residue. Such fragments, also referred to aspeptides or polypeptides, may contain antigenic regions, functionalregions of the protein identified as regions of the amino acid sequencewhich correspond to known protein domains, as well as regions ofpronounced hydrophilicity. The regions are all easily identifiable byusing commonly available protein sequence analysis software such asMacVector (Oxford Molecular).

Contemplated variants further include those containing predeterminedmutations by, e.g., homologous recombination, site-directed or PCRmutagenesis, and the corresponding proteins of other animal species,including but not limited to rabbit, mouse, rat, porcine, bovine, ovine,equine and non-human primate species, and the alleles or other naturallyoccurring variants of the families of proteins (for example, a mousehomolog that shows similarity to the mouse protein corresponding toGenBank Accession No. XM_(—)128002, XM_(—)129365, NM_(—)021420,NM_(—)133971 (DNA sequence) and NP_(—)598732 (protein sequence), all ofwhich are incorporated herein by reference.) Additional variants includederivatives wherein the protein has been covalently modified bysubstitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid (for example adetectable moiety such as an enzyme or radioisotope).

The present invention further provides compositions comprising a proteinor polypeptide of the invention and a diluent. Suitable diluents can beaqueous or non-aqueous solvents or a combination thereof, and cancomprise additional components, for example water-soluble salts orglycerol, that contribute to the stability, solubility, activity, and/orstorage of the protein or polypeptide.

As described below, members of the families of proteins can be used: (1)to identify agents which modulate the level of or at least one activityof the protein, (2) to identify binding partners for the protein, (3) asan antigen to raise polyclonal or monoclonal antibodies, (4) as atherapeutic agent or target and (5) as a diagnostic agent or marker ofstomach cancer and other hyperplastic diseases.

B. Nucleic Acid Molecules

The present invention further provides nucleic acid molecules thatencode the protein having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18 andthe related proteins herein described, preferably in isolated form. Asused herein, “nucleic acid” is defined as RNA or DNA that encodes aprotein or peptide as defined above; is complementary to a nucleic acidsequence encoding such peptides; hybridizes to the nucleic acid of SEQID NO: 1, 3, 5, 7, 9, 11, 13 or 17 and remains stably bound to it underappropriate stringency conditions; encodes a polypeptide sharing atleast about 50%, 60%, 70% or 75%, preferably at least about 80-90%, morepreferably at least about 92-95%, and most preferably at least about95-98% or more identity with the peptide sequence of SEQ ID NO: 2 or 4;exhibits at least 50%, 60%, 70% or 75%, preferably at least about80-90%, more preferably at least about 92-95%, and even more preferablyat least about 95-98% or more nucleotide sequence identity over the openreading frames of SEQ ID NO: 1 or 3; encodes a polypeptide sharing atleast about 50%, 60%, 70% or 75%, preferably at least about 80%, morepreferably at least about 85%, and most preferably at least about 90%,95%, 98%, 99%, 99.5% or more identity with the peptide sequence of SEQID NO: 6, 8, 10 or 12; exhibits at least 50%, 60%, 70% or 75%,preferably at least about 80%, more preferably at least about 85%, andeven more preferably at least about 90%, 95%, 98%, 99%, 99.5% or morenucleotide sequence identity over the open reading frames of SEQ ID NO:5, 7, 9 or 11; encodes a polypeptide sharing at least about 50%, 60%,70% or 75%, preferably at least about 80-90%, more preferably at leastabout 92-94%, and most preferably at least about 95%, 98%, 99% or moreidentity with the peptide sequence of SEQ ID NO: 14 or 18; or exhibitsat least 50%, 60%, 70% or 75%, preferably at least about 80-90%, morepreferably at least about 92-94%, and even more preferably at leastabout 95%, 98%, 99% or more nucleotide sequence identity over the openreading frame of SEQ ID NO: 13 or 17.

The present invention further includes isolated nucleic acid moleculesthat specifically hybridize to the complement of SEQ ID NO: 1, 3, 5, 7,9, 11, 13 or 17, particularly molecules that specifically hybridize overthe open reading frame. Such molecules that specifically hybridize tothe complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17 typically do sounder stringent hybridization conditions.

Specifically contemplated are genomic DNA, cDNA, mRNA and antisensemolecules, as well as nucleic acids based on alternative backbones orincluding alternative bases, whether derived from natural sources orsynthesized. Such hybridizing or complementary nucleic acids, however,are defined further as being novel and unobvious over any prior artnucleic acid including that which encodes, hybridizes under appropriatestringency conditions, or is complementary to nucleic acid encoding aprotein according to the present invention.

Homology or identity at the nucleotide or amino acid sequence level isdetermined by BLAST (Basic Local Alignment Search Tool) analysis usingthe algorithm employed by the programs blastp, blastn, blastx, tblastnand tblastx (Altschul et al., (1997) Nucleic Acids Res 25:3389-3402, andKarlin et al., (1990) Proc Natl Acad Sci USA 87:2264-2268, both fullyincorporated by reference) which are tailored for sequence similaritysearching. The approach used by the BLAST program is to first considersimilar segments, with and without gaps, between a query sequence and adatabase sequence, then to evaluate the statistical significance of allmatches that are identified and finally to summarize only those matcheswhich satisfy a preselected threshold of significance. For a discussionof basic issues in similarity searching of sequence databases, seeAltschul et al., (1994)

Nature Genetics 6: 119-129 which is fully incorporated by reference. Thesearch parameters for histogram, descriptions, alignments, expect (i.e.,the statistical significance threshold for reporting matches againstdatabase sequences), cutoff, matrix and filter (low complexity) are atthe default settings. The default scoring matrix used by blastp, blastx,tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992)Proc Natl Acad Sci USA 89:10915-10919, fully incorporated by reference),recommended for query sequences over 85 nucleotides or amino acids inlength.

For blastn, the scoring matrix is set by the ratios of M (i.e., thereward score for a pair of matching residues) to N (i.e., the penaltyscore for mismatching residues), wherein the default values for M and Nare 5 and −4, respectively. Four blastn parameters were adjusted asfollows: Q=10 (gap creation penalty); R=10 (gap extension penalty);wink=1 (generates word hits at every wink^(th) position along thequery); and gapw-16 (sets the window width within which gappedalignments are generated). The equivalent Blastp parameter settings wereQ=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences,available in the GCG package version 10.0, uses DNA parameters GAP=50(gap creation penalty) and LEN=3 (gap extension penalty) and theequivalent settings in protein comparisons are GAP=8 and LEN=2.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is hybridization in 50% formamide,5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicatedsalmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C., with washes at 42° C. in 0.2×SSC and 0.1% SDS. A skilled artisan canreadily determine and vary the stringency conditions appropriately toobtain a clear and detectable hybridization signal. Preferred moleculesare those that hybridize under the above conditions to the complement ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17 and which encode a functional orfull-length protein. Even more preferred hybridizing molecules are thosethat hybridize under the above conditions to the complement strand ofthe open reading frame of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules encoding other polypeptides.

The present invention further provides fragments of the disclosednucleic acid molecules. As used herein, a fragment of a nucleic acidmolecule refers to a small portion of the coding or non-coding sequence.The size of the fragment will be determined by the intended use. Forexample, if the fragment is chosen so as to encode an active portion ofthe protein, the fragment will need to be large enough to encode thefunctional region(s) of the protein. For instance, fragments whichencode peptides corresponding to predicted antigenic regions may beprepared. If the fragment is to be used as a nucleic acid probe or PCRprimer, then the fragment length is chosen so as to obtain a relativelysmall number of false positives during probing/priming (see thediscussion in Section H).

Fragments of the nucleic acid molecules of the present invention (i.e.,synthetic oligonucleotides) that are used as probes or specific primersfor the polymerase chain reaction (PCR), or to synthesize gene sequencesencoding proteins of the invention, can easily be synthesized bychemical techniques, for example, the phosphoramidite method ofMatteucci et al., ((1981) J Am Chem Soc 103:3185-3191) or usingautomated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the gene,followed by ligation of oligonucleotides to build the complete modifiedgene.

The nucleic acid molecules of the present invention may further bemodified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled orfluorescently labeled nucleotides and the like. A skilled artisan canreadily employ any such label to obtain labeled variants of the nucleicacid molecules of the invention.

C. Isolation of Other Related Nucleic Acid Molecules

As described above, the identification and characterization of thenucleic acid molecule having SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17allows a skilled artisan to isolate nucleic acid molecules that encodeother members of the protein families in addition to the sequencesherein described. Further, the presently disclosed nucleic acidmolecules allow a skilled artisan to isolate nucleic acid molecules thatencode other members of the families of proteins in addition to theproteins having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18.

For instance, a skilled artisan can readily use the amino acid sequenceof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18 to generate antibody probesto screen expression libraries prepared from appropriate cells.Typically, polyclonal antiserum from mammals such as rabbits immunizedwith the purified protein (as described below) or monoclonal antibodiescan be used to probe a mammalian cDNA or genomic expression library,such as lambda gt11 library, to obtain the appropriate coding sequencefor other members of the protein families. The cloned cDNA sequence canbe expressed as a fusion protein, expressed directly using its owncontrol sequences, or expressed by constructions using control sequencesappropriate to the particular host used for expression of the enzyme.

Alternatively, a portion of the coding sequence herein described can besynthesized and used as a probe to retrieve DNA encoding a member of theprotein family from any mammalian organism. Oligomers containingapproximately 18-20 nucleotides (encoding about a 6-7 amino acidstretch) are prepared and used to screen genomic DNA or cDNA librariesto obtain hybridization under stringent conditions or conditions ofsufficient stringency to eliminate an undue level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for usein a polymerase chain reaction (PCR) to selectively clone an encodingnucleic acid molecule. A PCR denature/anneal/extend cycle for using suchPCR primers is well known in the art and can readily be adapted for usein isolating other encoding nucleic acid molecules.

Nucleic acid molecules encoding other members of the protein familiesmay also be identified in existing genomic or other sequence informationusing any available computational method, including but not limited to:PSI-BLAST (Altschul et al., (1997) Nucleic Acids Res 25:3389-3402);PHI-BLAST (Zhang et al., (1998) Nucleic Acids Res 26:3986-3990), 3D-PSSM(Kelly et al., (2000) J Mol Biol 299(2):499-520); and othercomputational analysis methods (Shi et al., (1999) Biochem Biophys ResCommun 262(1):132-138 and Matsunami et. al., (2000) Nature404(6778):601-604.

D. rDNA Molecules Containing a Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs)that contain a coding sequence. As used herein, a rDNA molecule is a DNAmolecule that has been subjected to molecular manipulation in situ.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning—A Laboratory Manual,Third Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001. In the preferred rDNA molecules, a coding DNA sequence isoperably linked to expression control sequences and/or vector sequences.

The choice of vector and/or expression control sequences to which one ofthe protein family encoding sequences of the present invention isoperably linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed. A vector contemplated by the present inventionis at least capable of directing the replication or insertion into thehost chromosome, and preferably also expression, of the structural geneincluded in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, and other regulatory elements. Preferably,the inducible promoter is readily controlled, such as being responsiveto a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid moleculewill include a prokaryotic replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extrachromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such replicons arewell known in the art. In addition, vectors that include a prokaryoticreplicon may also include a gene whose expression confers a detectablemarker such as a drug resistance. Typical bacterial drug resistancegenes are those that confer resistance to ampicillin, kanamycin,chloramphenicol or tetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from BioRad Laboratories, (Richmond,Calif.), pPL and pKK223 available from Pharmacia (Piscataway, N.J.).

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, such as stomach cells, can also beused to form rDNA molecules that contain a coding sequence. Eukaryoticcell expression vectors, including viral vectors, are well known in theart and are available from several commercial sources. Typically, suchvectors are provided containing convenient restriction sites forinsertion of the desired DNA segment. Typical of such vectors are pSVLand pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies,Inc.), pTDT1 (ATCC, #31255), the vector pCDM8 described herein, and thelike eukaryotic expression vectors. Vectors may be modified to includestomach cell specific promoters if needed.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. (Southern et al., (1982) J Mol Anal Genet 1:327-341)Alternatively, the selectable marker can be present on a separateplasmid, and the two vectors are introduced by co-transfection of thehost cell, and selected by culturing in the appropriate drug for theselectable marker.

E. Host Cells Containing an Exogenously Supplied Coding Nucleic AcidMolecule

The present invention further provides host cells transformed with anucleic acid molecule that encodes a protein of the present invention.The host cell can be either prokaryotic or eukaryotic. Eukaryotic cellsuseful for expression of a protein of the invention are not limited, solong as the cell line is compatible with cell culture methods andcompatible with the propagation of the expression vector and expressionof the gene product. Preferred eukaryotic host cells include, but arenot limited to, yeast, insect and mammalian cells, preferably vertebratecells such as those from a mouse, rat, monkey or human cell line.Preferred eukaryotic host cells include Chinese hamster ovary (CHO)cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells(NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells(BHK), and the like eukaryotic tissue culture cell lines.

Any prokaryotic host can be used to express a rDNA molecule encoding aprotein of the invention. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed (see, for example, Cohen etal., (1972) Proc Natl Acad Sci USA 69:2110; and Sambrook et al., supra).With regard to transformation of vertebrate cells with vectorscontaining rDNAs, electroporation, cationic lipid or salt treatmentmethods are typically employed, see, for example, Graham et al., (1973)Virol 52:456; Wigler et al., (1979) Proc Natl Acad Sci USA 76;1373-1376.

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniquesincluding the selection for a selectable marker. For example, cellsresulting from the introduction of an rDNA of the present invention canbe cloned to produce single colonies. Cells from those colonies can beharvested, lysed and their DNA content examined for the presence of therDNA using a method such as that described by Southern, (1975) J MolBiol 98:503 or Berent et al., (1985) Biotech 3:208, or the proteinsproduced from the cell assayed via an immunological method.

F. Production of Recombinant Proteins Using a rDNA Molecule

The present invention further provides methods for producing a proteinof the invention using nucleic acid molecules herein described. Ingeneral terms, the production of a recombinant form of a proteintypically involves the following steps:

First, a nucleic acid molecule is obtained that encodes a protein of theinvention, such as a nucleic acid molecule comprising, consistingessentially of or consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:17, nucleotides 131-862 or 131-859 of SEQ ID NO: 1, nucleotides 174-587or 174-584 of SEQ ID NO: 3, nucleotides 38-892 or 38-895 of SEQ ID NO:5, nucleotides 53-892 or 53-895 of SEQ ID NO: 7, nucleotides 65-892 or65-895 of SEQ ID NO: 9, or nucleotides 92-892 or 92-895 of SEQ ID NO:11, nucleotides 49-1437 or 49-1434 of SEQ ID NO: 13, or nucleotides75-575 or 75-572 of SEQ ID NO: 17. If the encoding sequence isuninterrupted by introns, as are these open-reading-frames, it isdirectly suitable for expression in any host.

The nucleic acid molecule is then preferably placed in operable linkagewith suitable control sequences, as described above, to form anexpression unit containing the protein open reading frame. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of therecombinant protein. Optionally the recombinant protein is isolated fromthe medium or from the cells; recovery and purification of the proteinmay not be necessary in some instances where some impurities may betolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with the nucleic acid molecules of theinvention to produce recombinant protein.

G. Methods to Identify Binding Partners

Another embodiment of the present invention provides methods forisolating and identifying binding partners of proteins of the invention.In general, a protein of the invention is mixed with a potential bindingpartner or an extract or fraction of a cell under conditions that allowthe association of potential binding partners with the protein of theinvention. After mixing, peptides, polypeptides, proteins or othermolecules that have become associated with a protein of the inventionare separated from the mixture. The binding partner that bound to theprotein of the invention can then be removed and further analyzed. Toidentify and isolate a binding partner, the entire protein, for instancea protein comprising the entire amino acid sequence of SEQ ID NO: 2, 4,6, 8, 10, 12, 14 or 18 can be used. Alternatively, a fragment of theprotein can be used.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell. The preferred source ofcellular extracts will be cells derived from human stomach tumors ortransformed stomach cells, for instance, biopsy tissue or tissue culturecells from gastric carcinomas. Alternatively, cellular extracts may beprepared from normal tissue or available cell lines, particularlystomach-derived cell lines.

A variety of methods can be used to obtain an extract of a cell. Cellscan be disrupted using either physical or chemical disruption methods.Examples of physical disruption methods include, but are not limited to,sonication and mechanical shearing. Examples of chemical lysis methodsinclude, but are not limited to, detergent lysis and enzyme lysis. Askilled artisan can readily adapt methods for preparing cellularextracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with theprotein of the invention under conditions in which association of theprotein with the binding partner can occur. A variety of conditions canbe used, the most preferred being conditions that closely resembleconditions found in the cytoplasm of a human cell. Features such asosmolarity, pH, temperature, and the concentration of cellular extractused, can be varied to optimize the association of the protein with thebinding partner.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to a protein ofthe invention can be used to immunoprecipitate the binding partnercomplex. Alternatively, standard chemical separation techniques such aschromatography and density/sediment centrifugation can be used.

After removal of non-associated cellular constituents found in theextract, the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the protein of the invention can be immobilized on a solidsupport. For example, the protein can be attached to a nitrocellulosematrix or acrylic beads. Attachment of the protein to a solid supportaids in separating peptide/binding partner pairs from other constituentsfound in the extract. The identified binding partners can be either asingle protein or a complex made up of two or more proteins.Alternatively, binding partners may be identified using a Far-Westernassay according to the procedures of Takayama et al., (1997) Methods MolBiol 69:171-184 or Sauder et al., (1996) J Gen Virol 77:991-996 oridentified through the use of epitope tagged proteins or GST fusionproteins.

Alternatively, the nucleic acid molecules of the invention can be usedin a yeast two-hybrid system or other in vivo protein-protein detectionsystem. The yeast two-hybrid system has been used to identify otherprotein partner pairs and can readily be adapted to employ the nucleicacid molecules herein described.

H. Methods to Identify Agents that Modulate the Expression a NucleicAcid Encoding the Genes Associated with Stomach Cancer

Another embodiment of the present invention provides methods foridentifying agents that modulate the expression of a nucleic acidencoding a protein of the invention such as a protein having the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 18, or a Mst1 proteinor splice variant of the invention such as a protein having the aminoacid sequence of SEQ ID NO: 14. The agents that modulate the expressionof the nucleic acid encoding the Mst1 protein or splice variant willhave particular use in the treatment of stomach cancer. Such assays mayutilize any available means of monitoring for changes in the expressionlevel of the nucleic acids of the invention. As used herein, an agent issaid to modulate the expression of a nucleic acid of the invention if itis capable of up- or down-regulating expression of the nucleic acid in acell.

In one assay format, cell lines that contain reporter gene fusionsbetween nucleotides from within the open reading frame defined bynucleotides 131-862 of SEQ ID NO: 1, or nucleotides 174-587 of SEQ IDNO: 3, nucleotides 38-895 of SEQ ID NO: 5, nucleotides 53-895 of SEQ IDNO: 7, nucleotides 65-895 of SEQ ID NO: 9, nucleotides 92-895 of SEQ IDNO: 11, nucleotides 49-1437 or 49-1434 of SEQ ID NO: 13, nucleotides75-575 of SEQ ID NO: 17 and/or the 5′ and/or 3′ regulatory elements andany assayable fusion partner may be prepared. Numerous assayable fusionpartners are known and readily available including the fireflyluciferase gene and the gene encoding chloramphenicol acetyltransferase(Alam et al., (1990) Anal Biochem 188:245-254). Cell lines containingthe reporter gene fusions are then exposed to the agent to be testedunder appropriate conditions and time. Differential expression of thereporter gene between samples exposed to the agent and control samplesidentifies agents which modulate the expression of a nucleic acid of theinvention.

Additional assay formats may be used to monitor the ability of the agentto modulate the expression of a nucleic acid encoding a protein of theinvention, such as the protein having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14or 18. For instance, mRNA expression may be monitored directly byhybridization to the nucleic acids of the invention. Cell lines areexposed to the agent to be tested under appropriate conditions and timeand total RNA or mRNA is isolated by standard procedures such thosedisclosed in Sambrook et al., Molecular Cloning—A Laboratory Manual,Third Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001.

The preferred cells will be those derived from human stomach tissue, forinstance, stomach biopsy tissue or cultured cells from patients withstomach cancer. Cell lines such as ATCC gastric carcinoma cell lineCatalogue Nos. NCI-SNU-16, CRL-1863, HTB-103, CRL-1739 and CRL-1864 maybe used. Alternatively, other available cells or cell lines may be used.

Probes to detect differences in RNA expression levels between cellsexposed to the agent and control cells may be prepared from the nucleicacids of the invention. It is preferable, but not necessary, to designprobes which hybridize only with target nucleic acids under conditionsof high stringency. Only highly complementary nucleic acid hybrids formunder conditions of high stringency. Accordingly, the stringency of theassay conditions determines the amount of complementarity which shouldexist between two nucleic acid strands in order to form a hybrid.Stringency should be chosen to maximize the difference in stabilitybetween the probe:target hybrid and probe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention throughmethods known in the art. For instance, the G+C content of the probe andthe probe length can affect probe binding to its target sequence.Methods to optimize probe specificity are commonly available in Sambrooket al., supra, or Ausubel et al., Short Protocols in Molecular Biology,Fourth Ed., John Wiley & Sons, Inc., New York, 1999.

Hybridization conditions are modified using known methods, such as thosedescribed by Sambrook et al. and Ausubel et al. as required for eachprobe. Hybridization of total cellular RNA or RNA enriched for polyA RNAcan be accomplished in any available format. For instance, totalcellular RNA or RNA enriched for polyA RNA can be affixed to a solidsupport and the solid support exposed to at least one probe comprisingat least one, or part of one of the sequences of the invention underconditions in which the probe will specifically hybridize.Alternatively, nucleic acid fragments comprising at least one, or partof one of the sequences of the invention can be affixed to a solidsupport, such as a silicon chip, porous glass wafer or membrane. Thesolid support can then be exposed to total cellular RNA or polyA RNAfrom a sample under conditions in which the affixed sequences willspecifically hybridize. Such solid supports and hybridization methodsare widely available, for example, those disclosed by Beattie, (1995) WO95/11755. By examining for the ability of a given probe to specificallyhybridize to an RNA sample from an untreated cell population and from acell population exposed to the agent, agents which up- or down-regulatethe expression of a nucleic acid encoding the protein having thesequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18 are identified.

Hybridization for qualitative and quantitative analysis of mRNAs mayalso be carried out by using a RNase Protection Assay (i.e., RPA, see Maet al., (1996) Methods 10:273-238). Briefly, an expression vehiclecomprising cDNA encoding the gene product and a phage specific DNAdependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase)is linearized at the 3′ end of the cDNA molecule, downstream from thephage promoter, wherein such a linearized molecule is subsequently usedas a template for synthesis of a labeled antisense transcript of thecDNA by in vitro transcription. The labeled transcript is thenhybridized to a mixture of isolated RNA (i.e., total or fractionatedmRNA) by incubation at 45° C. overnight in a buffer comprising 80%formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resultinghybrids are then digested in a buffer comprising 40 μg/ml ribonuclease Aand 2 μg/ml ribonuclease. After deactivation and extraction ofextraneous proteins, the samples are loaded onto urea/polyacrylamidegels for analysis.

In another assay, to identify agents which affect the expression of theinstant gene products, cells or cell lines are first identified whichexpress the gene products of the invention physiologically. Cell and/orcell lines so identified would be expected to comprise the necessarycellular machinery such that the fidelity of modulation of thetranscriptional apparatus is maintained with regard to exogenous contactof agent with appropriate surface transduction mechanisms and/or thecytosolic cascades. Further, such cells or cell lines would betransduced or transfected with an expression vehicle (e.g., a plasmid orviral vector) construct comprising an operable non-translated5′promoter-containing end of the structural gene encoding the instantgene products fused to one or more antigenic fragments, which arepeculiar to the instant gene products, wherein said fragments are underthe transcriptional control of said promoter and are expressed aspolypeptides whose molecular weight can be distinguished from thenaturally occurring polypeptides or may further comprise animmunologically distinct tag or other detectable marker. Such a processis well known in the art (see Sambrook et al., supra).

Cells or cell lines transduced or transfected as outlined above are thencontacted with agents under appropriate conditions. For example, theagent in a pharmaceutically acceptable excipient is contacted with cellsin an aqueous physiological buffer such as phosphate buffered saline(PBS) at physiological pH, Eagles balanced salt solution (BSS) atphysiological pH, PBS or BSS comprising serum or conditioned mediacomprising PBS or BSS and/or serum incubated at 37° C. Said conditionsmay be modulated as deemed necessary by one of skill in the art.Subsequent to contacting the cells with the agent, said cells will bedisrupted and the polypeptides of the lysate are fractionated such thata polypeptide fraction is pooled and contacted with an antibody to befurther processed by immunological assay (e.g., ELISA,immunoprecipitation or Western blot). The pool of proteins isolated fromthe “agent-contacted” sample will be compared with a control samplewhere only the excipient is contacted with the cells and an increase ordecrease in the immunologically generated signal from the“agent-contacted” sample compared to the control will be used todistinguish the effectiveness of the agent.

I. Methods to Identify Agents that Modulate the Level or at Least OneActivity of the Stomach Cancer Associated Proteins

Another embodiment of the present invention provides methods foridentifying agents that modulate the level or at least one activity of aprotein of the invention such as the protein having the amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 18, or of a Mst1 protein orsplice variant of the invention such as the protein having the aminoacid sequence of SEQ ID NO: 14. Such methods or assays may utilize anymeans of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the inventionbetween a cell population that has been exposed to the agent to betested compared to an un-exposed control cell population may be assayed.In this format, probes such as specific antibodies are used to monitorthe differential expression of the protein in the different cellpopulations. Cell lines or populations are exposed to the agent to betested under appropriate conditions and time. Cellular lysates may beprepared from the exposed cell line or population and a control,unexposed cell line or population. The cellular lysates are thenanalyzed with the probe.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides, polypeptides orproteins of the invention if they are of sufficient length, or, ifdesired, or if required to enhance immunogenicity, conjugated tosuitable carriers. Methods for preparing immunogenic conjugates withcarriers such as BSA, KLH, or other carrier proteins are well known inthe art. In some circumstances, direct conjugation using, for example,carbodiimide reagents may be effective; in other instances linkingreagents such as those supplied by Pierce Chemical Co. (Rockford, Ill.),may be desirable to provide accessibility to the hapten. The haptenpeptides can be extended at either the amino or carboxy terminus with acysteine residue or interspersed with cysteine residues, for example, tofacilitate linking to a carrier. Administration of the immunogens isconducted generally by injection over a suitable time period and withuse of suitable adjuvants, as is generally understood in the art. Duringthe immunization schedule, titers of antibodies are taken to determineadequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein ((1975) Nature 256:495-497) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe peptide hapten, polypeptide or protein. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in ascitesfluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of the monoclonalantibodies or the polyclonal antisera which contain the immunologicallysignificant (antigen-binding) portion can be used as antagonists, aswell as the intact antibodies. Use of immunologically reactive(antigen-binding) antibody fragments, such as the Fab, Fab′, or F(ab′)₂fragments is often preferable, especially in a therapeutic context, asthese fragments are generally less immunogenic than the wholeimmunoglobulin.

The antibodies or antigen-binding fragments may also be produced, usingcurrent technology, by recombinant means. Antibody regions that bindspecifically to the desired regions of the protein can also be producedin the context of chimeras with multiple species origin, such ashumanized antibodies.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences involved in the association of a protein of theinvention alone or with its associated substrates, binding partners,etc. An example of randomly selected agents is the use a chemicallibrary or a peptide combinatorial library, or a growth broth of anorganism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that make up these sites.For example, a rationally selected peptide agent can be a peptide whoseamino acid sequence is identical to or a derivative of any functionalconsensus site.

The agents of the present invention can be, as examples, peptides, smallmolecules, vitamin derivatives, as well as carbohydrates. Dominantnegative proteins, DNAs encoding these proteins, antibodies to theseproteins, peptide fragments of these proteins or mimics of theseproteins may be introduced into cells to affect function. “Mimic” usedherein refers to the modification of a region or several regions of apeptide molecule to provide a structure chemically different from theparent peptide but topographically and functionally similar to theparent peptide (see Grant in: Molecular Biology and Biotechnology,Meyers, ed., pp. 659-664, VCH Publishers, Inc., New York, 1995). Askilled artisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solidphase (or solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of proteins of the invention.Antibody agents are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the protein intended to be targeted by the antibodies.

J. Uses for Agents that Modulate the Expression or at Least One Activityof the Proteins Associated with Stomach Cancer

As provided in the Examples, the proteins and nucleic acids of theinvention, such as the proteins having the amino acid sequence of SEQ IDNO: 2, 4, 6, 8, 10, 12 or 18, and the Mst1 or Mst1 splice variantproteins and nucleic acids of the invention, such as the proteins havingthe amino acid sequence of SEQ ID NO: 14 are differentially expressed incancerous stomach tissue. Agents that up- or down-regulate or modulatethe expression of the protein or at least one activity of the protein,such as agonists or antagonists, of may be used to modulate biologicaland pathologic processes associated with the protein's function andactivity.

For example, two types of drugs have been shown to act through Mst1(e.g., GenBank Accession No. NM_(—)006282, the nucleic acid and proteinsequences for which are given as SEQ ID NOS: 15 and 16, respectively), agene related to SEQ ID NOS: 13 and 14. Firstly, it has been shown thatbisphophonates, drugs that are used to treat osteoporosis and other bonediseases, act directly on the osteoclast to induce caspase cleavage ofMst1 during apoptosis. Secondly, cytotrienin A is an antitumor drug thatis used to treat leukemia, breast cancer and lung cancer (U.S. Pat. No.6,251,885). Cytotrienin A has been shown to activate Mst1 duringcytotrienin A-induced apoptosis (Watabe et al., (2000) J Biol Chem275:8766-8771).

As used herein, a subject can be any mammal, so long as the mammal is inneed of modulation of a pathological or biological process mediated by aprotein of the invention. The term “mammal” is defined as an individualbelonging to the class Mammalia. The invention is particularly useful inthe treatment of human subjects.

Pathological processes refer to a category of biological processes whichproduce a deleterious effect. For example, expression of a protein ofthe invention may be associated with stomach cell growth or hyperplasia.As used herein, an agent is said to modulate a pathological process whenthe agent reduces the degree or severity of the process. For instance,stomach cancer may be prevented or disease progression modulated by theadministration of agents which up- or down-regulate or modulate in someway the expression or at least one activity of a protein of theinvention.

The agents of the present invention can be provided alone, or incombination with other agents that modulate a particular pathologicalprocess. For example, an agent of the present invention can beadministered in combination with other known drugs. As used herein, twoagents are said to be administered in combination when the two agentsare administered simultaneously or are administered independently in afashion such that the agents will act at the same time.

The agents of the present invention can be administered via parenteral,subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal,or buccal routes. Alternatively, or concurrently, administration may beby the oral route. The dosage administered will be dependent upon theage, health, and weight of the recipient, kind of concurrent treatment,if any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one ormore agents which modulate expression or at least one activity of aprotein of the invention. While individual needs vary, determination ofoptimal ranges of effective amounts of each component is within theskill of the art. Typical dosages comprise 0.1 to 100 μg/kg body wt. Thepreferred dosages comprise 0.1 to 10 μg/kg body wt. The most preferreddosages comprise 0.1 to 1 μg/kg body wt.

In addition to the pharmacologically active agent, the compositions ofthe present invention may contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically for delivery to the site of action. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form, for example, water-solublesalts. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers. Liposomes can also be used to encapsulate the agentfor delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of thisinvention may be used alone or in combination, or in combination withother therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadininisteredalong with other compounds typically prescribed for these conditionsaccording to generally accepted medical practice. The compounds of thisinvention can be utilized in vivo, ordinarily in mammals, such ashumans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or invitro.

K. Transgenic Animals

Transgenic animals containing mutant, knock-out or modified genescorresponding to the cDNA sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13or 17, or the open reading frame encoding the polypeptide sequence ofSEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 18 or fragments thereof having aconsecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30,35 or more amino acid residues, are also included in the invention.Transgenic animals are genetically modified animals into whichrecombinant, exogenous or cloned genetic material has beenexperimentally transferred. Such genetic material is often referred toas a “transgene.” The nucleic acid sequence of the transgene, in thiscase a form of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17 may be integratedeither at a locus of a genome where that particular nucleic acidsequence is not otherwise normally found or at the normal locus for thetransgene. The transgene may consist of nucleic acid sequences derivedfrom the genome of the same species or of a different species than thespecies of the target animal.

In some embodiments, transgenic animals in which all or a portion of agene comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17 is deleted may beconstructed. In those cases where the gene corresponding to SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 17 contains one or more introns, the entiregene—all exons, introns and the regulatory sequences—may be deleted.Alternatively, less than the entire gene may be deleted. For example, asingle exon and/or intron may be deleted, so as to create an animalexpressing a modified version of a protein of the invention.

The term “germ cell line transgenic animal” refers to a transgenicanimal in which the genetic alteration or genetic information wasintroduced into a germ line cell, thereby conferring the ability of thetransgenic animal to transfer the genetic information to offspring. Ifsuch offspring in fact possess some or all of that alteration or geneticinformation, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species ofanimal to which the recipient belongs, foreign only to the particularindividual recipient, or may be genetic information already possessed bythe recipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

Transgenic animals can be produced by a variety of different methodsincluding transfection, electroporation, microinjection, gene targetingin embryonic stem cells and recombinant viral and retroviral infection(see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins etal., (1993) Hypertension 22:630-633; Brenin et al., (1997) Surg Oncol6:99-110; Recombinant Gene Expression Protocols (Methods in MolecularBiology, Vol. 62), Tuan, ed., Humana Press, Totowa, N.J., 1997).

A number of recombinant or transgenic mice have been produced, includingthose which express an activated oncogene sequence (U.S. Pat. No.4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915);lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat.No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No.5,723,719); express at least one human gene which participates in bloodpressure control (U.S. Pat. No. 5,731,489); display greater similarityto the conditions existing in naturally occurring Alzheimer's disease(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellularadhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene(Clutter et al., (1996) Genetics 143:1753-1760); or, are capable ofgenerating a fully human antibody response (McCarthy (1997) Lancet349:405).

While mice and rats remain the animals of choice for most transgenicexperimentation, in some instances it is preferable or even necessary touse alternative animal species. Transgenic procedures have beensuccessfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see, e.g., Kim et al., (1997) Mol Reprod Dev46:515-526; Houdebine, (1995) Reprod Nutr Dev 35:609-617; Petters (1994)Reprod Fertil Dev 6:643-645; Schnieke et al., (1997) Science278:2130-2133; and Amoah, (1997) J Animal Science 75:578-585).

The method of introduction of nucleic acid fragments into recombinationcompetent mammalian cells can be by any method which favorsco-transformation of multiple nucleic acid molecules. Detailedprocedures for producing transgenic animals are readily available to oneskilled in the art, including the disclosures in U.S. Pat. No. 5,489,743and U.S. Pat. No. 5,602,307.

L. Diagnostic Methods

As the genes and proteins of the invention are differentially expressedin cancerous stomach tissue and in other malignant neoplasms compared tonon-cancerous tissues of the same type, the genes and proteins of theinvention may be used to diagnose or monitor such cancers or to trackdisease progression. One means of diagnosing cancer, including stomachcancer, using the nucleic acid molecules or proteins of the inventioninvolves obtaining tissue from living subjects, such as biopsyspecimens.

The use of molecular biological tools has become routine in forensictechnology. For example, nucleic acid probes comprising all or at leastpart of the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17 may beused to determine the expression of a nucleic acid molecule inforensic/pathology specimens. Further, nucleic acid assays may becarried out by any means of conducting a transcriptional profilinganalysis. In addition to nucleic acid analysis, forensic methods of theinvention may target the proteins of the invention, particularly aprotein comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, todetermine up- or down-regulation of the genes (Shiverick et al., (1975)Biochim Biophys Acta 393:124-133).

Methods of the invention may involve treatment of tissues withcollagenases or other proteases to make the tissue amenable to celllysis (Semenov et al., (1987) Biull Eksp Biol Med 104:113-116). Further,it is possible to obtain biopsy samples from different regions of thestomach for analysis.

Assays to detect nucleic acid or protein molecules of the invention maybe in any available format. Typical assays for nucleic acid moleculesinclude hybridization or PCR based formats. Typical assays for thedetection of proteins, polypeptides or peptides of the invention includethe use of antibody probes in any available format such as in situbinding assays, etc. (see Harlow & Lane, Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988. Inpreferred embodiments, assays are carried-out with appropriate controls.

The above methods may also be used in other diagnostic protocols,including protocols and methods to detect disease states in othertissues or organs, for example in tissues in which expression of anucleic acid molecule of the invention is detected.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES Example 1a

Identification of Differentially Expressed mRNA in Advanced GastricCarcinoma

Materials and Methods

Patient tissue samples were derived from five Korean patients, aged 47to 68, including four men and one woman, who had been diagnosed withadvanced gastric cancer. For each patient, tissue was obtained from twoareas of the stomach, from a stomach tumor and from a cancer-free area,to produce a set of biopsy samples. Histological analysis of each of thetissue samples was performed, and samples were segregated into eithernon-cancerous or cancerous categories.

With minor modifications, the sample preparation protocol followed theAffymetrix GeneChip Expression Analysis Manual. Frozen tissue was firstground to powder using the Spex Certiprep 6800 Freezer Mill. Total RNAwas then extracted using Trizol (Life Technologies). The total RNA yieldfor each sample (average tissue weight of 300 mg) was 200-500 μg. Next,mRNA was isolated using the Oligotex mRNA Midi kit (Qiagen). Since themRNA was eluted in a final volume of 400 μl, an ethanol precipitationstep was required to bring the concentration to 1 μg/μl. Using 1-5 μg ofmRNA, double stranded cDNA was created using the SuperScript Choicesystem (Gibco-BRL). First strand cDNA synthesis was primed with aT7-(dT₂₄) oligonucleotide. The cDNA was then phenol-chloroform extractedand ethanol precipitated to a final concentration of 1 μg/μl

From 2 μg of cDNA, cRNA was synthesized according to standardprocedures. To biotin label the cRNA, nucleotides Bio-11-CTP andBio-16-UTP (Enzo Diagnostics) were added to the reaction. After a 37° C.incubation for six hours, the labeled cRNA was cleaned up according tothe RNeasy Mini kit protocol (Qiagen). The cRNA was then fragmented (5×fragmentation buffer: 200 mM Tris-Acetate (pH 8.1), 500 mM KOAc, 150 mMMgOAc) for thirty-five minutes at 94° C.

55 μg of fragmented cRNA was hybridized on the Affymetrix Human GenomeU95 and U133 set of arrays for twenty-four hours at 60 rpm in a 45° C.hybridization oven. The chips were washed and stained with StreptavidinPhycoerythrin (SAPE) (Molecular Probes) in Affymetrix fluidics stations.To amplify staining, SAPE solution was added twice with ananti-streptavidin biotinylated antibody (Vector Laboratories) stainingstep in between. Hybridization to the probe arrays was detected byfluorometric scanning (Hewlett Packard Gene Array Scanner). Followinghybridization and scanning, the microarray images were analyzed forquality control, looking for major chip defects or abnormalities inhybridization signal. After all chips passed QC, the data was analyzedusing Affymetrix Microarray Suite (v4.0), and LIMS (v1.5) for U95 orAffymetrix Microarray Suite (v5.0), and LIMS (v3.0) for U133.

Differential expression of genes between the cancerous and non-cancerousliver samples was determined by using Affymetrix human GeneChip sets,U95 and U133, with the following statistical methods. (1) For each gene,Affymetrix GeneChip average difference values for U95 were determined byAffymetrix Microarray Suite (v4.0), which also made “Absent” (=notdetected), “Tresent” (=detected) or “Marginal” (=not clearly Absent orPresent) calls for each GeneChip element. Signal values for U133 weredetermined by Affymetrix Microarray Suite (v5.0), which also madeAbsent, Present or Marginal calls. (2) Using the criteria of at least10% present call in both cancerous and non-cancerous liver samples andat least 40% present call in either cancerous or non-cancerous liversample groups, a gene set was selected for further analysis. (3) Basedon the average difference values of U95 data, the gene set was splitinto two groups, a high expression group and low expression group. Thehigh expression group contained genes with average difference valuesgreater than or equal to 5 in both cancerous and non-cancerous samples.The remainder of the genes were included in the low expression group.The average difference values were transformed to a logarithmic scalefor the high expression group, but were not changed for the lowexpression group. For U133 data, all signal values were transformed to alogarithmic scale regardless of expression level. (4) The Analysis ofVariance (ANOVA) method was used for data analysis (Steel et al.,Principles and Procedures of Statistics: A Biometrical Approach, ThirdEd., McGraw-Hill, 1997). Prior to the final analysis, a leave-one-outapproach is used for outlier detection. One sample at a time was leftout of the ANOVA analysis to determine whether or not omitting aspecific sample from the analysis had any significant effect on thefinal result. If so, that particular sample was excluded from the finalanalysis. After outlier detection, a list of genes that aredifferentially expressed with a p-value of less than or equal to 0.05was generated by ANOVA. Data from Affymetrix GeneChip U133 chip set wasanalyzed with a similar procedure. (5) Two additional criteria were usedto reduce the number of genes in the gene list generated from U95.Firstly, geometric mean values were compared between the non-cancerouscontrol group samples and the carcinoma disease group samples to obtaina set of genes showing at least 2.0-fold increases or decreases inexpression level. Secondly, the ratio of the fold-change value and thep-value had to be 400 or greater.

Results and Analyses

a) LBFL301 Gene Family:

Analysis of the chip data showed that the expression of the markerLBFL301 was significantly up-regulated (13.75-fold; p=0.0172) in gastriccarcinoma samples compared to samples from normal stomach tissue. Thesedata indicate that up-regulation of LBFL301 may be diagnostic forstomach cancer.

The expression level of LBFL301 (SEQ ID NO: 1 or 3) can be measured bychip sequence fragment nos. 48774_at and 225681_at on AffymetrixGeneChips® U95 and U133, respectively. The expression levels of 48774_atand 225681_at in various malignant neoplasms, compared to normal controltissues, are shown in Table 1a, where the fold-change and the directionof the change (up- or down-regulation) are also indicated. A fold-changegreater than 1.5 was considered to be significant. TABLE 1a Affy IDTissue Disease Morphology Fold Change Dir T-Stat 48774_at BONES, NOSMALIGNANT NEOPLASM OF BONE, NOS GIANT CELL TUMOR OF BONE, NOS 5.7 Up 3.5225681_at BREAST, NOS MALIGNANT NEOPLASM OF FEMALE BREAST, NOSINFILTRATING DUCT CARCINOMA 3.4 Up 12.1 225681_at INFILTRATING DUCT ANDLOBULAR CARCINOMA 3.3 Up 3.3 225681_at INFILTRATING LOBULAR CARCINOMA2.8 Up 5.3 225681_at CERVIX, NOS MALIGNANT NEOPLASM OF UTERINE CERVIXSQUAMOUS CELL CARCINOMA, NOS 3.6 Up 3.4 225681_at COLON, NOS MALIGNANTNEOPLASM OF COLON, NOS MUCINOUS ADENOCARCINOMA 12.7 Up 2.5 225681_atADENOCARCINOMA, NOS 6.9 Up 7.9 225681_at ENDOMETRIUM, NOS MALIGNANTNEOPLASM OF ENDOMETRIUM MULLERIAN MIXED TUMOR 11.0 Up 5.5 225681_atADENOCARCINOMA, NOS 2.2 Up 3.7 225681_at ESOPHAGUS, NOS MALIGNANTNEOPLASM OF ESOPHAGUS, NOS ADENOCARCINOMA, NOS 7.2 Up 3.6 225681_atKIDNEY, NOS MALIGNANT NEOPLASM OF KIDNEY, NOS CLEAR CELL ADENOCARCINOMA,NOS 9.8 Up 5.4 225681_at WILMS' TUMOR 7.2 Up 3.4 225681_at LARYNX, NOSMALIGNANT NEOPLASM OF LARYNX, NOS SQUAMOUS CELL CARCINOMA, NOS 6.6 Up4.3 225681_at LIVER, NOS SECONDARY MALIGNANT NEOPLASM OF LIVER, NOSADENOCARCINOMA, NOS 12.4 Up 3.5 225681_at LUNG, NOS MALIGNANT NEOPLASMOF LUNG, NOS SQUAMOUS CELL CARCINOMA, NOS 5.4 Up 5.4 225681_atADENOCARCINOMA, NOS 4.4 Up 5.1 225681_at LYMPH NODE, NOS SECONDARYMALIGNANT NEOPLASM OF LYMPH NODE, NOS SQUAMOUS CELL CARCINOMA, NOS 4.1Up 2.7 225681_at MALIGNANT NEOPLASM OF LYMPHOID AND HISTIOCYTICMALIGNANT LYMPHOMA, NOS −4.3 Down −3.2 TISSUE, NOS 225681_at OMENTUM,NOS MALIGNANT NEOPLASM OF THE OMENTUM PAPILLARY SEROUS ADENOCARCINOMA7.2 Up 4.1 225681_at SECONDARY MALIGNANT NEOPLASM OF THE OMENTUMPAPILLARY SEROUS ADENOCARCINOMA 4.8 Up 4.3 225681_at MULLERIAN MIXEDTUMOR 4.7 Up 11.0 225681_at OVARY, NOS MALIGNANT NEOPLASM OF OVARYADENOCARCINOMA, NOS 13.8 Up 2.6 225681_at SEROUS CYSTADENOCARCINOMA, NOS10.7 Up 2.8 225681_at NEOPLASM OF UNCERTAIN BEHAVIOR OF OVARY STRUMAOVARII, NOS 9.5 Up 6.4 225681_at MALIGNANT NEOPLASM OF OVARY PAPILLARYSEROUS ADENOCARCINOMA 7.7 Up 3.7 225681_at SECONDARY MALIGNANT NEOPLASMOF OVARY ADENOCARCINOMA, NOS 6.9 Up 3.7 225681_at NEOPLASM OF UNCERTAINBEHAVIOR OF OVARY SEROUS CYSTADENOMA, BORDERLINE 5.3 Up 7.2 MALIGNANCY225681_at PANCREAS, NOS MALIGNANT NEOPLASM OF PANCREAS, NOSADENOCARCINOMA, NOS 7.8 Up 9.3 225681_at RECTUM, NOS MALIGNANT NEOPLASMOF RECTUM ADENOCARCINOMA, NOS 7.7 Up 5.8 225681_at SOFT TISSUES, NOSNEOPLASM OF UNCERTAIN BEHAVIOR OF CONNECTIVE AND FIBROMATOSIS, NOS 12.5Up 7.6 OTHER SOFT TISSUES, NOS 225681_at SECONDARY MALIGNANT NEOPLASM OFCONNECTIVE AND SQUAMOUS CELL CARCINOMA, NOS 5.3 Up 3.2 OTHER SOFTTISSUES, NOS 225681_at MALIGNANT NEOPLASM OF CONNECTIVE AND OTHER SOFTFIBROUS HISTIOCYTOMA, MALIGNANT 3.7 Up 3.1 TISSUES, NOS 225681_atSTOMACH, NOS MALIGNANT NEOPLASM OF STOMACH, NOS ADENOCARCINOMA, NOS 3.6Up 3.5 225681_at SIGNET RING CELL CARCINOMA 3.5 Up 4.5 225681_at THYROIDGLAND, MALIGNANT NEOPLASM OF THYROID GLAND PAPILLARY CARCINOMA, NOS 4.5Up 4.1 NOS

Table 2 summarizes the differential expression data collected fromexperiments using Affymetrix GeneChips by tissue type. The chips werescanned and the data analyzed by the GX Scan algorithm, which isdescribed in related applications 60/331,182, 60/388,745 and 60/390,608,all entitled “An Automated Computer-based Algorithm for Organizing andMining Gene Expression Data Derived from Biological Samples with ComplexClinical Attributes,” and all of which are herein incorporated byreference in their entirety. TABLE 2 LBFL301 (U95: 48774_at, U133:225681_at): Clones AD12 & CH4 48774_at 225681_at From U95 data From U133data 1. Bone UP — 2. Breast UP UP 3. Cervix UP UP 4. Colon UP UP 5.Endometrium UP UP 6. Esophagus UP UP 7. Kidney UP UP 8. Larynx UP UP 9.Liver UP UP 10. Lung UP UP 11. Omentum UP UP 12. Ovary UP UP 13.Pancreas UP UP 14. Rectum UP UP 15. Soft tissues UP UP 16. Stomach UP UP17. Thyroid Gland UP UP

The GeneChip expression results, determined by sample binding to chipsequence fragment no. 48774_at were validated by quantitative RT-PCRusing Taqman® assay (Perkin-Elmer). PCR primers designed from the sifsequence of the specific Affymetrix fragment (48774_at) were used in theassay. The target gene in each RNA sample (ten ng of total RNA) wasassayed relative to an exogenously spiked reference gene. For thispurpose, the tetracycline resistance gene was used as the exogenouslyadded spike. This approach provides the relative expression as measuredby cycle threshold (Ct) value of the target mRNA relative to a constantamount of Tet spike Ct values. The sample panel included normal andadvanced gastric cancer tissue RNAs that were analyzed on U95 GeneChips.In addition, several new samples that were not analyzed on the GeneChipwere used for the expression validations by Quantitative RT-PCR. TheQ-RT-PCR data confirms the up-regulation of LBFL301 observed in advancedgastric cancer.

b) LBFL304 Gene Family:

Analysis of the chip data showed that the expression of the markerLBFL304 was significantly up-regulated (3.5-fold, p=2.54×10⁻³ for U95;6.13-fold, p=2.43×10⁻⁴ for U133) in AGC samples compared to samples fromnormal stomach tissue. This data indicates that up-regulation of LBFL304may be diagnostic for stomach cancer.

The expression level of LBFL304 (SEQ ID NO: 5, 7, 9 or 11) can bemeasured by chip sequence fragment nos. 35832_at on AffymetrixGeneChips® U95 and 212344_at, 212353_at, and 212354_at on AffymetrixGeneChips® U133. The expression levels of 51263_at, 212344_at,212353_at, and 212354_at in various malignant neoplasms, compared tonormal control tissues, are shown in Table 1b, where the fold-change andthe direction of the change (up- or down-regulation) are also indicated.A fold-change greater than 1.5 was considered to be significant.

The GeneChip expression results, determined by sample binding to chipsequence fragment no. 35832_at, were validated by quantitative RT-PCR(Q-RT-PCR) using the Taqman® assay (Perkin-Elmer). PCR primers designedfrom the sequence information file of the specific Affymetrix fragment(35832_at) were used in the assay. The target gene in each RNA sample(10 ng of total RNA) was assayed relative to an exogenously spikedreference gene. For this purpose, the tetracycline resistance gene wasused as the exogenously added spike. This approach provides the relativeexpression as measured by cycle threshold (Ct) value of the target mRNArelative to a constant amount of Tet spike Ct values. The sample panelincluded normal stomach (Normal) and advanced gastric cancer (AGC)tissue RNAs that were analyzed on U95 GeneChips. In addition, severalnew samples that were not analyzed on the GeneChip were used for theexpression validations by Q-RT-PCR The Q-RT-PCR data confirms theup-regulation of LBFL304 observed in AGC, compared to normal stomachbiopsy samples. TABLE 1 Table 1b Fold Affy ID Tissue Disease MorphologyChange Dir T-Stat 212344_at BONES, NOS MALIGNANT NEOPLASM OF BONE, GIANTCELL TUMOR OF BONE, NOS 6.8 Up 6.9 NOS 212353_at MALIGNANT NEOPLASM OFBONE, GIANT CELL TUMOR OF BONE, NOS 5.5 Up 5.7 NOS 212354_at MALIGNANTNEOPLASM OF BONE, GIANT CELL TUMOR OF BONE, NOS 5.9 Up 7.1 NOS 35832_atMALIGNANT NEOPLASM OF BONE, GIANT CELL TUMOR OF BONE, NOS 10.6 Up 5.7NOS 212344_at COLON, NOS MALIGNANT NEOPLASM OF MUCINOUS ADENOCARCINOMA3.7 Up 2.8 COLON, NOS 212344_at MALIGNANT NEOPLASM OF ADENOCARCINOMA,NOS 2.7 Up 7.9 COLON, NOS 212353_at MALIGNANT NEOPLASM OF MUCINOUSADENOCARCINOMA 5.7 Up 3.7 COLON, NOS 212353_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 4.1 Up 9.0 COLON, NOS 212354_at MALIGNANT NEOPLASMOF MUCINOUS ADENOCARCINOMA 4.7 Up 3.5 COLON, NOS 212354_at MALIGNANTNEOPLASM OF ADENOCARCINOMA, NOS 3.0 Up 8.6 COLON, NOS 35832_at MALIGNANTNEOPLASM OF MUCINOUS ADENOCARCINOMA 9.4 Up 3.8 COLON, NOS 35832_atMALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 6.5 Up 10.2 COLON, NOS212344_at SOFT MALIGNANT NEOPLASM OF FIBROUS HISTIOCYTOMA, 2.7 Up 3.2TISSUES, CONNECTIVE AND OTHER SOFT MALIGNANT NOS TISSUES, NOS 212353_atMALIGNANT NEOPLASM OF FIBROUS HISTIOCYTOMA, 2.1 Up 2.2 CONNECTIVE ANDOTHER SOFT MALIGNANT TISSUES, NOS 212353_at MALIGNANT NEOPLASM OF MYXOIDLIPOSARCOMA −2.7 Down −2.4 CONNECTIVE AND OTHER SOFT TISSUES, NOS212354_at MALIGNANT NEOPLASM OF FIBROUS HISTIOCYTOMA, 2.2 Up 2.6CONNECTIVE AND OTHER SOFT MALIGNANT TISSUES, NOS 35832_at MALIGNANTNEOPLASM OF FIBROUS HISTIOCYTOMA, 3.5 Up 2.6 CONNECTIVE AND OTHER SOFTMALIGNANT TISSUES, NOS 35832_at MALIGNANT NEOPLASM OF LIPOSARCOMA, NOS4.8 Up 2.6 CONNECTIVE AND OTHER SOFT TISSUES, NOS 212344_at ENDOMETRIUM,MALIGNANT NEOPLASM OF MULLERIAN MIXED TUMOR 2.4 Up 3.3 NOS ENDOMETRIUM212344_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 2.2 Up 5.5ENDOMETRIUM 212353_at MALIGNANT NEOPLASM OF MULLERIAN MIXED TUMOR 1.9 Up2.9 ENDOMETRIUM 212353_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 2.0Up 4.2 ENDOMETRIUM 212354_at MALIGNANT NEOPLASM OF MULLERIAN MIXED TUMOR2.0 Up 2.7 ENDOMETRIUM 212354_at MALIGNANT NEOPLASM OF ADENOCARCINOMA,NOS 1.9 Up 4.7 ENDOMETRIUM 35832_at MALIGNANT NEOPLASM OF MULLERIANMIXED TUMOR 3.0 Up 3.9 ENDOMETRIUM 35832_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 2.4 Up 4.7 ENDOMETRIUM 212344_at ESOPHAGUS,MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 1.7 Up 2.3 NOS ESOPHAGUS, NOS212353_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 5.5 Up 3.2ESOPHAGUS, NOS 212354_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 2.9Up 3.1 ESOPHAGUS, NOS 35832_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS10.7 Up 3.2 ESOPHAGUS, NOS 212344_at BREAST, MALIGNANT NEOPLASM OFMUCINOUS ADENOCARCINOMA 3.3 Up 4.8 NOS FEMALE BREAST, NOS 212344_atMALIGNANT NEOPLASM OF INFILTRATING LOBULAR 1.6 Up 2.9 FEMALE BREAST, NOSCARCINOMA 212344_at MALIGNANT NEOPLASM OF INFILTRATING DUCT CARCINOMA2.5 Up 8.7 FEMALE BREAST, NOS 212344_at MALIGNANT NEOPLASM OFINFILTRATING DUCT AND LOBULAR 2.6 Up 2.8 FEMALE BREAST, NOS CARCINOMA212353_at MALIGNANT NEOPLASM OF INFILTRATING LOBULAR 2.8 Up 4.7 FEMALEBREAST, NOS CARCINOMA 212353_at MALIGNANT NEOPLASM OF INFILTRATING DUCTCARCINOMA 3.9 Up 13.0 FEMALE BREAST, NOS 212353_at MALIGNANT NEOPLASM OFINFILTRATING DUCT AND LOBULAR 3.4 Up 3.7 FEMALE BREAST, NOS CARCINOMA212354_at MALIGNANT NEOPLASM OF INFILTRATING LOBULAR 1.7 Up 3.1 FEMALEBREAST, NOS CARCINOMA 212354_at MALIGNANT NEOPLASM OF INFILTRATING DUCTCARCINOMA 2.6 Up 8.5 FEMALE BREAST, NOS 212354_at MALIGNANT NEOPLASM OFINFILTRATING DUCT AND LOBULAR 2.7 Up 2.6 FEMALE BREAST, NOS CARCINOMA35832_at MALIGNANT NEOPLASM OF MUCINOUS ADENOCARCINOMA 4.0 Up 5.5 FEMALEBREAST, NOS 35832_at MALIGNANT NEOPLASM OF INFILTRATING LOBULAR 3.2 Up4.4 FEMALE BREAST, NOS CARCINOMA 35832_at MALIGNANT NEOPLASM OFINFILTRATING DUCT CARCINOMA 4.5 Up 12.0 FEMALE BREAST, NOS 35832_atMALIGNANT NEOPLASM OF INFILTRATING DUCT AND LOBULAR 4.3 Up 4.5 FEMALEBREAST, NOS CARCINOMA 212353_at KIDNEY, NOS MALIGNANT NEOPLASM OF CLEARCELL ADENOCARCINOMA, 2.8 Up 5.5 KIDNEY, NOS NOS 212354_at MALIGNANTNEOPLASM OF CLEAR CELL ADENOCARCINOMA, 2.4 Up 5.3 KIDNEY, NOS NOS35832_at MALIGNANT NEOPLASM OF CLEAR CELL ADENOCARCINOMA, 6.1 Up 5.6KIDNEY, NOS NOS 212344_at LARYNX, MALIGNANT NEOPLASM OF SQUAMOUS CELLCARCINOMA, 3.0 Up 3.3 NOS LARYNX, NOS NOS 212353_at MALIGNANT NEOPLASMOF SQUAMOUS CELL CARCINOMA, 5.1 Up 3.8 LARYNX, NOS NOS 212354_atMALIGNANT NEOPLASM OF SQUAMOUS CELL CARCINOMA, 3.7 Up 3.6 LARYNX, NOSNOS 35832_at MALIGNANT NEOPLASM OF SQUAMOUS CELL CARCINOMA, 7.2 Up 3.7LARYNX, NOS NOS 212344_at LUNG, NOS MALIGNANT NEOPLASM OF LUNG,CARCINOMA, NOS 4.1 Up 3.9 NOS 212344_at MALIGNANT NEOPLASM OF LUNG,SQUAMOUS CELL CARCINOMA, 3.1 Up 5.8 NOS NOS 212344_at MALIGNANT NEOPLASMOF LUNG, LARGE CELL CARCINOMA, NOS 2.7 Up 2.5 NOS 212344_at MALIGNANTNEOPLASM OF LUNG, ADENOCARCINOMA, NOS 2.8 Up 6.2 NOS 212353_at MALIGNANTNEOPLASM OF LUNG, CARCINOMA, NOS 4.1 Up 3.2 NOS 212353_at MALIGNANTNEOPLASM OF LUNG, SQUAMOUS CELL CARCINOMA, 3.6 Up 6.5 NOS NOS 212353_atMALIGNANT NEOPLASM OF LUNG, LARGE CELL CARCINOMA, NOS 3.8 Up 2.7 NOS212353_at MALIGNANT NEOPLASM OF LUNG, ADENOCARCINOMA, NOS 3.6 Up 6.9 NOS212354_at MALIGNANT NEOPLASM OF LUNG, CARCINOMA, NOS 3.5 Up 2.9 NOS212354_at MALIGNANT NEOPLASM OF LUNG, SQUAMOUS CELL CARCINOMA, 3.1 Up5.6 NOS NOS 212354_at MALIGNANT NEOPLASM OF LUNG, LARGE CELL CARCINOMA,NOS 2.9 Up 2.6 NOS 212354_at MALIGNANT NEOPLASM OF LUNG, ADENOCARCINOMA,NOS 2.9 Up 6.2 NOS 35832_at MALIGNANT NEOPLASM OF LUNG, SQUAMOUS CELLCARCINOMA, 8.8 Up 6.0 NOS NOS 35832_at MALIGNANT NEOPLASM OF LUNG,ADENOCARCINOMA, NOS 8.9 Up 7.2 NOS 212344_at OVARY, NOS MALIGNANTNEOPLASM OF OVARY PAPILLARY SEROUS 3.0 Up 3.9 ADENOCARCINOMA 212353_atMALIGNANT NEOPLASM OF OVARY PAPILLARY SEROUS 3.6 Up 3.9 ADENOCARCINOMA212354_at MALIGNANT NEOPLASM OF OVARY PAPILLARY SEROUS 2.6 Up 3.4ADENOCARCINOMA 212344_at PANCREAS, MALIGNANT NEOPLASM OF ADENOCARCINOMA,NOS 6.9 Up 9.0 NOS PANCREAS, NOS 212353_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 7.8 Up 11.2 PANCREAS, NOS 212354_at MALIGNANTNEOPLASM OF ADENOCARCINOMA, NOS 8.6 Up 11.0 PANCREAS, NOS 35832_atMALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 99.8 Up 11.1 PANCREAS, NOS212353_at PROSTATE, MALIGNANT NEOPLASM OF ATYPIA SUSPICIOUS FOR −2.2Down −4.7 NOS PROSTATE MALIGNANCY 212353_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS −1.5 Down −2.5 PROSTATE 212354_at MALIGNANT NEOPLASMOF ATYPIA SUSPICIOUS FOR −1.8 Down −3.8 PROSTATE MALIGNANCY 212354_atMALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS −1.7 Down −3.8 PROSTATE35832_at MALIGNANT NEOPLASM OF ATYPIA SUSPICIOUS FOR −4.3 Down −4.4PROSTATE MALIGNANCY 35832_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS−1.7 Down −2.6 PROSTATE 212344_at RECTUM, MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 3.0 Up 6.9 NOS RECTUM 212353_at MALIGNANT NEOPLASMOF ADENOCARCINOMA, NOS 4.1 Up 7.6 RECTUM 212354_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 3.0 Up 7.2 RECTUM 35832_at MALIGNANT NEOPLASM OFADENOCARCINOMA, NOS 5.4 Up 8.8 RECTUM 212344_at SKIN, NOS MALIGNANTNEOPLASM OF SKIN, SQUAMOUS CELL CARCINOMA, 2.8 Up 2.4 NOS NOS 212353_atMALIGNANT NEOPLASM OF SKIN, SQUAMOUS CELL CARCINOMA, 3.5 Up 2.4 NOS NOS35832_at MALIGNANT NEOPLASM OF SKIN, BASAL CELL CARCINOMA, NOS 3.6 Up2.5 NOS 35832_at MALIGNANT NEOPLASM OF SKIN, SQUAMOUS CELL CARCINOMA,5.5 Up 3.0 NOS NOS 212344_at STOMACH, MALIGNANT NEOPLASM OF MUCINOUSADENOCARCINOMA 7.6 Up 3.1 NOS STOMACH, NOS 212344_at MALIGNANT NEOPLASMOF SIGNET RING CELL CARCINOMA 3.9 Up 2.4 STOMACH, NOS 212344_atMALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 2.5 Up 4.9 STOMACH, NOS212353_at MALIGNANT NEOPLASM OF MUCINOUS ADENOCARCINOMA 11.0 Up 3.7STOMACH, NOS 212353_at MALIGNANT NEOPLASM OF SIGNET RING CELL CARCINOMA4.9 Up 2.9 STOMACH, NOS 212353_at MALIGNANT NEOPLASM OF ADENOCARCINOMA,NOS 3.4 Up 5.6 STOMACH, NOS 212354_at MALIGNANT NEOPLASM OF MUCINOUSADENOCARCINOMA 7.5 Up 3.2 STOMACH, NOS 212354_at MALIGNANT NEOPLASM OFSIGNET RING CELL CARCINOMA 3.7 Up 2.4 STOMACH, NOS 212354_at MALIGNANTNEOPLASM OF ADENOCARCINOMA, NOS 2.7 Up 5.1 STOMACH, NOS 35832_atMALIGNANT NEOPLASM OF SIGNET RING CELL CARCINOMA 8.4 Up 3.4 STOMACH, NOS35832_at MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 8.6 Up 6.7 STOMACH,NOS 212344_at TESTIS, NOS MALIGNANT NEOPLASM OF MIXED GERM CELL TUMOR2.0 Up 2.4 TESTIS, NOS 212353_at MALIGNANT NEOPLASM OF MIXED GERM CELLTUMOR 4.1 Up 2.8 TESTIS, NOS 212354_at MALIGNANT NEOPLASM OF MIXED GERMCELL TUMOR 3.4 Up 2.6 TESTIS, NOS 212353_at OMENTUM, MALIGNANT NEOPLASMOF THE PAPILLARY SEROUS 1.7 Up 2.6 NOS OMENTUM ADENOCARCINOMA 35832_atTHYROID MALIGNANT NEOPLASM OF PAPILLARY CARCINOMA, NOS 2.5 Up 2.5 GLAND,NOS THYROID GLAND 35832_at CERVIX, NOS MALIGNANT NEOPLASM OF SQUAMOUSCELL CARCINOMA, 3.5 Up 2.3 UTERINE CERVIX NOSc) LBFL305 Gene Family:

Analysis of the chip data showed that the expression of the markerLBFL305 was significantly up-regulated (2.2-fold, p=0.0051 using the U95GeneChip; 2.14-fold, p=0.0109 using the U133 GeneChip) in gastriccarcinoma samples compared to samples from normal stomach tissue. Thesedata indicate that up-regulation of LBFL305 may be diagnostic forstomach cancer.

The expression level of LBFL305 (SEQ ID NO: 13) can be measured by chipsequence fragment nos. 53858_at and 225364_at on Affymetrix GeneChips®U95 and U133, respectively. Differential expression data were collectedfrom experiments using Affymetrix GeneChips® by tissue type and wereanalyzed by the GX Scan algorithm, which is described in relatedapplications 60/331,182, 60/388,745 and 60/390,608, all entitled “AnAutomated Computer-based Algorithm for Organizing and Mining GeneExpression Data Derived from Biological Samples with Complex ClinicalAttributes,” and all of which are herein incorporated by reference intheir entirety. The expression levels of 53858_at and 225364_at invarious malignant neoplasms, compared to normal control tissues, areshown in Table 1c, where the fold-change and the direction of the change(up- or down-regulation) are also indicated. A fold-change greater than1.5 was considered to be significant. TABLE 1c Affy ID Tissue DiseaseMorphology Fold Change Dir T-Stat 53858_at BLADDER, NOS MALIGNANTNEOPLASM OF BLADDER, NOS TRANSITIONAL CELL CARCINOMA, NOS 1.691 Up 2.37753858_at BREAST, NOS MALIGNANT NEOPLASM OF FEMALE BREAST, NOSINFILTRATING DUCT CARCINOMA 1.538 Up 6.824 53858_at MALIGNANT NEOPLASMOF FEMALE BREAST, NOS INFILTRATING DUCT & LOBULAR CARCINOMA 1.599 Up2.408 225364_at MALIGNANT NEOPLASM OF FEMALE BREAST, NOS INFILTRATINGDUCT & LOBULAR CARCINOMA 1.525 Up 3.251 53858_at CERVIX, NOS MALIGNANTNEOPLASM OF UTERINE CERVIX SQUAMOUS CELL CARCINOMA, NOS 1.637 Up 4.05653858_at ENDOMETRIUM, NOS MALIGNANT NEOPLASM OF ENDOMETRIUM MULLERIANMIXED TUMOR 1.548 Up 2.647 53858_at ESOPHAGUS, NOS MALIGNANT NEOPLASM OFESOPHAGUS, NOS ADENOCARCINOMA, NOS 2.079 Up 3.145 225364_at MALIGNANTNEOPLASM OF ESOPHAGUS, NOS ADENOCARCINOMA, NOS 1.615 Up 2.915 53858_atKIDNEY, NOS MALIGNANT NEOPLASM OF KIDNEY, NOS RENAL CELL CARCINOMA 1.864Up 6.093 53858_at MALIGNANT NEOPLASM OF KIDNEY, NOS CLEAR CELLADENOCARCINOMA, NOS 2.167 Up 6.877 225364_at MALIGNANT NEOPLASM OFKIDNEY, NOS RENAL CELL CARCINOMA 1.593 Up 5.045 225364_at MALIGNANTNEOPLASM OF KIDNEY, NOS CLEAR CELL ADENOCARCINOMA, NOS 1.683 Up 7.924225364_at LUNG, NOS MALIGNANT NEOPLASM OF LUNG, NOS SMALL CELLCARCINOMA, NOS −1.79 Down −7.071 53858_at LYMPH NODE, NOS HODGKIN'SDISEASE, NOS OF LYMPH NODES OF MULTIPLE SITES HODGKIN'S DISEASE, NOS1.539 Up 2.519 53858_at MALIGNANT NEOPLASM OF LYMPHOID AND HISTIOCYTICMALIGNANT LYMPHOMA, NOS 1.635 Up 3.243 TISSUE, NOS 53858_at PANCREAS,NOS MALIGNANT NEOPLASM OF ISLETS OF LANGERHANS ISLET CELL CARCINOMA3.845 Up 7.929 53858_at MALIGNANT NEOPLASM OF PANCREAS, NOSADENOCARCINOMA, NOS 4.171 Up 7.165 225364_at MALIGNANT NEOPLASM OFISLETS OF LANGERHANS ISLET CELL CARCINOMA 2.343 Up 3.551 225364_atMALIGNANT NEOPLASM OF PANCREAS, NOS ADENOCARCINOMA, NOS 2.278 Up 10.793225364_at PROSTATE, NOS MALIGNANT NEOPLASM OF PROSTATE ATYPIA SUSPICIOUSFOR MALIGNANCY −1.641 Down −4.8 53858_at RECTUM, NOS MALIGNANT NEOPLASMOF RECTUM ADENOCARCINOMA, NOS 1.935 Up 5.416 53858_at SMALL INTESTINE,NOS MALIGNANT LYMPHOMA, NOS OF UNSPECIFIED, MALIGNANT LYMPHOMA, NOS3.935 Up 3.535 EXTRANODAL OR SOLID ORGAN SITE 225364_at MALIGNANTLYMPHOMA, NOS OF UNSPECIFIED, MALIGNANT LYMPHOMA, NOS 3.024 Up 3.816EXTRANODAL OR SOLID ORGAN SITE 53858_at STOMACH, NOS MALIGNANT NEOPLASMOF STOMACH, NOS SIGNET RING CELL CARCINOMA 1.507 Up 2.46 53858_atMALIGNANT NEOPLASM OF STOMACH, NOS ADENOCARCINOMA, NOS 2.076 Up 5.928225364_at MALIGNANT NEOPLASM OF STOMACH, NOS SIGNET RING CELL CARCINOMA1.524 Up 3.076 225364_at MALIGNANT NEOPLASM OF STOMACH, NOSADENOCARCINOMA, NOS 1.637 Up 6.053 225364_at THYROID GLAND, NOSMALIGNANT LYMPHOMA, NOS OF UNSPECIFIED, MALIGNANT LYMPHOMA, NOS 4.371 Up9.429 EXTRANODAL OR SOLID ORGAN SITE 225364_at MALIGNANT NEOPLASM OFTHYROID GLAND PAPILLARY CARCINOMA, NOS 1.634 Up 3.22 53858_at VULVA, NOSMALIGNANT NEOPLASM OF VULVA, NOS SQUAMOUS CELL CARCINOMA, NOS 1.505 Up2.596

The GeneChip expression results, determined by sample binding to chipsequence fragment no. 53858_at were validated by quantitative RT-PCR(Q-RT-PCR) using Taqman® assay (Perkin-Elmer). PCR primers designed fromthe sequence information file for the specific Affymetrix fragment(53858_at) were used in the assay. The target gene in each RNA sample(ten ng of total RNA) was assayed relative to an exogenously spikedreference gene. For this purpose, the tetracycline resistance gene wasused as the exogenously added spike. This approach provides the relativeexpression as measured by cycle threshold (Ct) value of the target mRNArelative to a constant amount of Tet spike Ct values. The sample panelincluded normal and advanced gastric cancer tissue RNAs that wereanalyzed on U95 GeneChips. In addition, several new samples that werenot analyzed on the GeneChip were used for the expression validations byQ-RT-PCR. The Q-RT-PCR data confirms the up-regulation of LBFL305observed in advanced gastric cancer.

d) LBFL306 Gene Family

Analysis of the chip data showed that the expression of the markerLBFL306 was significantly up-regulated (3.27-fold, p=0.00217 using theU133 GeneChip) in gastric carcinoma samples compared to samples fromnormal stomach tissue. These data indicate that up-regulation of LBFL306may be diagnostic for stomach cancer.

The expression level of LBFL306 (SEQ ID NO: 17, 19 or 21) can bemeasured by chip sequence fragment nos. 57861_at and 223251_s_at onAffymetrix GeneChips® U95 and U133, respectively. Differentialexpression data were collected from experiments using AffymetrixGeneChips® by tissue type and were analyzed by the GX Scan algorithm,which is described in related applications 60/331,182, 60/388,745 and60/390,608, all entitled “An Automated Computer-based Algorithm forOrganizing and Mining Gene Expression Data Derived from BiologicalSamples with Complex Clinical Attributes,” and all of which are hereinincorporated by reference in their entirety. The expression levels of223251_at in various malignant neoplasms, compared to normal controltissues, are shown in Table 1d, where the fold-change and the directionof the change (up- or down-regulation) are also indicated. A fold-changegreater than 1.5 was considered to be significant. The data show thatexpression of LBFL306 is up-regulated in cancers of the bladder, colon,esophagus, kidney, omentum, pancreas, rectum and soft tissues, inaddition to cancer of the stomach, and that expression of this genefamily is down-regulated in cancers of the breast, endometrium and smallintestine.

The full length cDNA having SEQ ID NO: 17 or 19 or 21 was obtained byusing GeneTrapper® cDNA Positive Selection System Kits (Invitrogen). Theresulting cDNA was converted to double-stranded plasmid DNA, used totransform E. coli cells (DH10B), and the longest cDNA was screened.After positive selection was confirmed by PCR using gene-specificprimers, the cDNA clone was subjected to DNA sequencing.

Analysis by Northern blot was performed to determine the size of themRNA transcripts that correspond to LBFL306. Northern blots containingtotal RNAs from various human tissues were used (ClonTech H12), andLBFL306-GE2 (SEQ ID NO: 21) was radioactively labeled by the randomprimer method and used to probe the blots. The blots were hybridized inChurch and Gilbert buffer at 65° C. and washed with 0.1×SSC containing0.1% SDS at room temperature. The Northern blots show a singletranscript for this gene, which is approximately 1.5 kb in size. Thiscorresponds to the size of the insert in full-length clones, which isalso approximately 1.5 kb.

The GeneChip expression results, determined by sample binding to chipsequence fragment no. 223251_s_at were validated by quantitative RT-PCR(Q-RT-PCR) using Taqman® assay (Perkin-Elmer). PCR primers designed fromthe sequence information file for the specific Affymetrix fragment(223251_s_at) were used in the assay. The target gene in each RNA sample(ten ng of total RNA) was assayed relative to an exogenously spikedreference gene. For this purpose, the tetracycline resistance gene wasused as the exogenously added spike. This approach provides the relativeexpression as measured by cycle threshold (Ct) value of the target mRNArelative to a constant amount of Tet spike Ct values. The sample panelincluded normal and advanced gastric cancer tissue RNAs that wereanalyzed on U133 GeneChips. In addition, several new samples that werenot analyzed on the GeneChip were used for the expression validations byQ-RT-PCR The Q-RT-PCR data conforms the up-regulation of LBFL306observed in advanced gastric cancer. TABLE 1d Chip position Fold no.Tissue Disease Morphology change Dir T-Stat 223251_s_at BLADDER, NOSMALIGNANT NEOPLASM OF BLADDER, NOS TRANSITIONAL CELL 1.649 Up 2.572CARCINOMA, NOS 223251_s_at BREAST, NOS MALIGNANT NEOPLASM OF FEMALEINTRADUCTAL −1.701 Down −5.202 BREAST, NOS CARCINOMA, NOS 223251_s_atCOLON, NOS MALIGNANT NEOPLASM OF COLON, NOS ADENOCARCINOMA, NOS 2.651 Up8.164 223251_s_at ENDOMETRIUM, MALIGNANT NEOPLASM OF ENDOMETRIUMMULLERIAN MIXED −1.806 Down −2.367 NOS TUMOR 223251_s_at ESOPHAGUS,MALIGNANT NEOPLASM OF ADENOCARCINOMA, NOS 5.016 Up 4.269 NOS ESOPHAGUS,NOS 223251_s_at KIDNEY, NOS MALIGNANT NEOPLASM OF KIDNEY, NOSTRANSITIONAL CELL 1.755 Up 4.563 CARCINOMA, NOS 223251_s_at MALIGNANTNEOPLASM OF KIDNEY, NOS CLEAR CELL 1.611 Up 4.054 ADENOCARCINOMA, NOS223251_s_at MALIGNANT NEOPLASM OF KIDNEY, NOS WILMS' TUMOR 4.565 Up3.891 223251_s_at OMENTUM, NOS MALIGNANT NEOPLASM OF THE OMENTUMPAPILLARY SEROUS 1.762 Up 3.398 ADENOCARCINOMA 223251_s_at PANCREAS, NOSMALIGNANT NEOPLASM OF PANCREAS, NOS ADENOCARCINOMA, NOS 2.672 Up 7.13223251_s_at RECTUM, NOS MALIGNANT NEOPLASM OF RECTUM ADENOCARCINOMA, NOS2.482 Up 6.042 223251_s_at SMALL MALIGNANT NEOPLASM OF SMALL SARCOMA,NOS −1.944 Down −5.716 INTESTINE, NOS INTESTINE, NOS 223251_s_at SOFTTISSUES, MALIGNANT NEOPLASM OF CONNECTIVE MYXOID LIPOSARCOMA 1.878 Up3.894 NOS AND OTHER SOFT TISSUES, NOS 223251_s_at STOMACH, NOS MALIGNANTNEOPLASM OF STOMACH, NOS ADENOCARCINOMA, NOS 2.522 Up 5.101

Example 2

Cloning of Full Length human cDNAs (LBFL301, LBFL304, LBFL305 andLBFL306) Corresponding to Differentially Expressed mRNA Species

The full length cDNA having SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 17, 19 or21 was obtained by the oligo-pulling method. Briefly, a gene-specificoligo was designed based on the sequence of LBFL301, LBFL304, LBFL305 orLBFL306. The oligo was labeled with biotin and used to hybridize with 2μg of single strand plasmid DNA (cDNA recombinants) from a fullydifferentiated stomach adenocarcinoma library (NCI CGAP Gas 4) or alibrary prepared from Jurkat cells following the procedures of Sambrooket al. The hybridized cDNAs were separated by streptavidin-conjugatedbeads and eluted by heating. The eluted cDNA was converted to doublestrand plasmid DNA and used to transform E. coli cells (DH10B) and thelongest cDNA was screened. After positive selection was confirmed by PCRusing gene-specific primers, the cDNA clone was subjected to DNAsequencing.

The nucleotide sequence of the full-length human cDNAs corresponding tothe differentially regulated mRNA detected above is set forth in SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 17, 19 and 21. In SEQ ID NO 1, the cDNAcomprises 1272 base pairs (1255 base pairs and a polyA tail). In SEQ IDNO 3, the cDNA comprises 1355 base pairs (1334 base pairs and a polyAtail). There are several possible start codons for LBFL304, and they aredesignated in SEQ ID NOS: 5, 7, 9 and 11. The cDNA in SEQ ID NO: 13comprises 6405 base paris (6369 base pairs and a poly A tail). The cDNAcorresponding to SEQ ID NO: 17 comprises 1299 base pairs (1284 basepairs and a polyA tail). The cDNA corresponding to SEQ ID NO: 19comprises 2451 base pairs (2435 base pairs and a polyA tail). The cDNAcorresponding to SEQ ID NO: 21 comprises 1194 base pairs (1178 basepairs and a polyA tail).

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:1, at nucleotides 131-859 (131-862 including the stop codon), encodes aprotein of 243 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 1 is set forth in SEQ ID NO: 2.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:3, at nucleotides 174-584 (174-587 including the stop codon), encodes aprotein of 137 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 3 is set forth in SEQ ID NO: 4.The protein sequence of SEQ ID NO: 4 is identical to that of SEQ ID NO:2 for the first 124 amino acids, while the last 13 amino acids of SEQ IDNO: 4 are unique. As shown in FIG. 1, termination of the proteinsequence corresponding to SEQ ID NO: 4 is produced by a 45-bp insertionwhich introduces a stop codon in the open reading frame.

SEQ ID NOS: 2 and 4 are weakly similar to the chymotrypsin serineprotease family signature (S1) and the NUDIX hydrolase family signature.The chymotrypsin serine protease family signature (S1) contains threedomains, the third of which is absent in SEQ ID NO: 4. Additionally,both proteins contain a domain of collagen triple helix repeats.

FIGS. 2 and 3 show the results of a hydrophobicity analysis of the aminoacid sequence of SEQ ID NOS: 2 and 4. Hydrophilic regions may be used toproduce antigenic peptides, as described above. Both sequences havehydrophobic N-termini, approximately 30 amino acids in length, with themost hydrophobic portion peaking at around amino acid no. 20. Furtherprotein sequence-analysis by SPScan (GCG Wisconsin Package) reveals thatthe hydrophobic regions from amino acid positions 1-30 are likely to besecretory signal peptides.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:5, at nucleotides 38-892 (38-895 including the stop codon), encodes aprotein of 285 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 5 is set forth in SEQ ID NO: 6.SEQ ID NO: 6 is weakly similar to the chymotrypsin serine proteasefamily (S1) signature. FIG. 4 shows the results of a hydrophobicityanalysis of the amino acid sequence of SEQ ID NO: 6. Hydrophilic regionsmay be used to produce antigenic peptides, as described above.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:7, at nucleotides 53-892 (53-895 including the stop codon), encodes aprotein of 280 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 7 is set forth in SEQ ID NO: 8.The protein sequence of SEQ ID NO: 8 is identical to that of SEQ ID NO:6, except that SEQ ID NO: 8 lacks the first five amino acids at theN-terminus of SEQ ID NO: 6.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:9, at nucleotides 65-892 (65-895 including the stop codon), encodes aprotein of 276 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 9 is set forth in SEQ ID NO: 10.The protein sequence of SEQ ID NO: 10 is identical to that of SEQ ID NO:6, except that SEQ ID NO: 10 lacks the first nine amino acids at theN-terminus of SEQ ID NO: 6.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:11, at nucleotides 92-892 (92-895 including the stop codon), encodes aprotein of 267 amino acids. The amino acid sequence corresponding to apredicted protein encoded by SEQ ID NO: 11 is set forth in SEQ ID NO:12. The protein sequence of SEQ ID NO: 12 is identical to that of SEQ IDNO: 6, except that SEQ ID NO: 12 lacks the first 18 amino acids at theN-terminus of SEQ ID NO: 6.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:13, at nucleotides 49-1434 (49-1437 including the stop codon), encodes aprotein of 462 amino acids. The amino acid sequence corresponding to theprotein encoded by SEQ ID NO: 13 is set forth in SEQ ID NO: 14.

BLAST search results and a high level of homology between the twosequences suggest that LBFL305 is a splice variant of Mst1 (e.g., of SEQID NO: 16). The underlined amino acid residues of the alignment indicatethe differences between SEQ ID NO: 14 and SEQ ID NO: 18. Based onpublished studies of Mst1, SEQ ID NO: 14 contains a kinase domain (aminoacid positions 1-299) (Creasy et al., (1996) J Biol Chem271:21049-21053), followed by a regulatory domain which acts to regulatekinase function (amino acid positions 300-462) (Creasy et al., (1996) JBiol Chem 271:21049-21053). Also present are two caspase cleavage sites,between amino acid positions 326-327 and 349-350 (Graves et al., (2001)J Biol Chem 276:14909-14915), and one NES domain (amino acid positions361-370) (Ura et al., (2002) Proc Natl Acad Sci USA 98: 10148-10153).Compared to SEQ ID NO: 16, SEQ ID NO: 14 is missing the second NESdomain (amino acid positions 441-451 in SEQ ID NO: 16) (Ura et al.,(2002) Proc Natl Acad Sci USA 98: 10148-10153). Also, SEQ ID NO: 14 doesnot contain the multimerization domain (amino acid positions 431-487 inMst1) that is required for self-association (Creasy et al., (1996) JBiol Chem 271:21049-21053). Interestingly, the region in Mst1 that isrequired for its interaction with NORE, a putative Ras effector (aminoacid positions 449-487 in SEQ ID NO: 16) (Khokhlatchev et al., Curr Biol12:253-265), is absent in SEQ ID NO: 14.

FIG. 5 show the results of a hydrophobicity analysis of the amino acidsequence of SEQ ID NO: 14. Hydrophilic regions may be used to produceantigenic peptides, as described above.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:17, at nucleotides 75-572 (75-575 including the stop codon), encodes aprotein of 166 amino acids. The amino acid sequence corresponding to theprotein encoded by SEQ ID NO: 17 is set forth in SEQ ID NO: 18. FIG. 7shows the results of a hydrophobicity analysis of the amino acidsequence of SEQ ID NO: 18. Hydrophilic regions may be used to produceantigenic peptides, as described above.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:19, at nucleotides 78-1337 (78-1340 including the stop codon), encodes aprotein of 420 amino acids. The amino acid sequence corresponding to theprotein encoded by SEQ ID NO: 19 is set forth in SEQ ID NO: 20. FIG. 8shows the results of a hydrophobicity analysis of the amino acidsequence of SEQ ID NO: 20. Hydrophilic regions may be used to produceantigenic peptides, as described above.

An open reading frame within the cDNA nucleotide sequence of SEQ ID NO:19, at nucleotides 78-737 (78-740 including the stop codon), encodes aprotein of 220 amino acids. The amino acid sequence corresponding to theprotein encoded by SEQ ID NO: 21 is set forth in SEQ ID NO: 22. FIG. 9shows the results of a hydrophobicity analysis of the amino acidsequence of SEQ ID NO: 22. Hydrophilic regions may be used to produceantigenic peptides, as described above.

All three LBFL306 clones, EF3 (SEQ ID NO: 17), GC7 (SEQ ID NO: 19) andGE2 (SEQ ID NO: 21), contain multiple ankyrin repeats, as determined byhmmerpfam, using GCG Wisconsin Package software. The ankyrin repeats arefrom amino acid residues 57 to 89, 91 to 123 and 124 to 156 in EF3, GC7and GE2. In addition to these three ankyrin repeats, GC7 contains anadditional ankyrin repeat from residues 157 to 190.

Analysis by Northern blot was performed to determine the size of themRNA transcripts that correspond to LBFL301, LBFL304 and LBFL305.Northern blots containing total RNAs from various human tissues wereused (ClonTech), and clone CH4 (SEQ ID NO: 3), clone EA10 (SEQ ID NO: 5,7, 9 or 11) and LBFL305 (SEQ ID NO: 13) were radioactively labeled bythe random primer method and used to probe the blots. The blots werehybridized in Church and Gilbert buffer at 65° C. and washed with0.1×SSC containing 0.1% SDS at room temperature. The Northern blots showa single transcript for each gene, which is approximately 1.57 kb(BFL301), 2.6 kb (BFL304) and 7.95 kb (LBFL305) in size. Thesecorrespond to the sizes of the inserts in clone CH4 (1.355 kb), cloneEA10 (SEQ ID NO: 5, 7, 9 or 11), and LBFL305(6.5 kb). When the sequenceof clone AD 12 (SEQ ID NO: 1) was used as the probe, a transcript of1.44 kb was detected, which corresponds to the size of the insert, 1.272kb, in clone AD12.

To examine the expression of LBFL301, LBFL304, LBFL305 or LBFL306 invarious normal tissues, an electronic Northern blot (e-Northern) wasprepared as follows. Using the chips and the procedures in Example 1,mRNA from a panel of normal tissues, as listed in Table 3, washybridized to Affymetrix U95 human GeneChips. The results of theseexperiments is shown in Table 3. For each tissue type, the number ofsamples that are called present or absent are indicated, together withthe total number of samples in that sample set. In addition, the medianvalue and the 25^(th) and 75^(th) percentiles in each tissue type arelisted. Interestingly, although this gene is up-regulated in stomachcancer, expression of LBFL301 or LBFL304 could not be detected in mostnormal stomach samples. In addition, although LBFL305 and LBFL306 werefound in most normal stomach samples tested, the level of expression waslower than in most other normal tissues tested. This observationindicates that LBFL301, LBFL304, LBFL305 or LBFL306 may be used as adiagnostic agent or marker to detect or screen for stomach cancer, asdiscussed below. Expression levels of LBFL301 appeared to be highest inskin tissue, followed by placental, adipose, arterial, bladder, bone,breast and soft tissues. Lower levels of expression were detected inmost of the other tissues listed in Table 3a, although this gene was notdetected in the liver or in most areas of the brain and heart.Expression levels of LBFL304 appeared to be highest in the arteries,omentum, uterus, endometrium, myometrium, and prostate. Expressionlevels of LBFL305 appeared to be highest in organs of the immune system(white blood cells, lymph nodes, spleen and thymus gland) followed bysamples from the appendix, artery, bone and lung. Still lower levels ofexpression were detected in most of the other tissues listed in Table3c. Expression levels of LBFL306 appeared to be highest in organs of theimmune system (e.g., lymph nodes, spleen and thymus gland) and of thereproductive system (e.g., breast, endometrium, prostate and uterus).TABLE 3a e-Northern Data for 48774_at: LBFL301 Gene Expression in NormalTissues Global Present Freq. Tissue Present Absent Lower 25% MedianUpper 75% 0.5492 Adipose 29 of 33  4 of 33 130.90 200.78 302.98 AdrenalGland  1 of 12 11 of 12 −4.10 8.75 22.07 Appendix 1 of 3 2 of 3 21.5431.31 71.81 Artery 3 of 3 0 of 3 148.46 203.96 262.32 Bladder 6 of 7 1of 7 142.72 195.44 361.02 Bone 2 of 4 2 of 4 75.00 240.38 412.62 Breast74 of 82  8 of 82 104.43 222.98 506.27 Cerebellum 0 of 5 5 of 5 −7.51−6.91 0.94 Cervix 75 of 99 24 of 99 42.04 93.36 144.25 Colon  36 of 148112 of 148 1.75 12.16 29.05 Cortex Frontal Lobe 1 of 7 6 of 7 5.96 14.0718.05 Cortex Temporal Lobe 0 of 3 3 of 3 −0.59 4.13 4.66 Duodenum  9 of61 52 of 61 3.99 11.54 20.62 Endometrium 16 of 21  5 of 21 46.25 85.18113.55 Esophagus 14 of 27 13 of 27 17.91 42.08 81.58 Fallopian Tube 21of 51 30 of 51 7.97 20.39 33.96 GallBladder 4 of 8 4 of 8 16.22 80.97400.47 Heart 0 of 3 3 of 3 −3.80 6.00 11.57 Hippocampus 1 of 5 4 of 5−6.49 −0.18 7.81 Kidney 12 of 87 75 of 87 −14.80 −4.61 9.24 Larynx 4 of4 0 of 4 48.51 119.88 248.62 Left Atrium  64 of 141  77 of 141 8.1927.15 61.94 Left Ventricle  2 of 15 13 of 15 −7.49 7.08 13.24 Liver  0of 33 33 of 33 −15.13 −8.62 0.03 Lung 43 of 92 49 of 92 10.45 30.1463.21 Lymph Node  9 of 12  3 of 12 43.28 81.96 225.75 Muscles 19 of 3819 of 38 23.70 40.22 108.40 Myometrium  68 of 106  38 of 106 19.39 56.4299.78 Omentum 12 of 16  4 of 16 76.26 148.41 236.54 Ovary 26 of 75 49 of75 4.20 21.96 47.43 Pancreas  7 of 34 27 of 34 −12.61 0.83 17.69Placenta 5 of 5 0 of 5 284.63 361.07 414.51 Prostate  7 of 32 25 of 320.03 12.08 36.90 Rectum 17 of 44 27 of 44 3.23 12.57 37.41 Right Atrium 60 of 171 111 of 171 2.99 15.73 53.22 Right Ventricle  43 of 160 117 of160 1.85 16.64 39.58 Skin 56 of 59  3 of 59 321.45 906.78 1515.60 SmallIntestine 18 of 68 50 of 68 0.41 12.19 28.53 Soft Tissues 5 of 6 1 of 6148.50 202.33 794.03 Spleen  5 of 29 24 of 29 −3.61 3.04 12.46 Stomach15 of 45 30 of 45 7.73 18.66 50.97 Testis 3 of 5 2 of 5 14.11 27.3464.24 Thymus 19 of 71 52 of 71 4.06 25.61 40.45 Thyroid Gland  7 of 1912 of 19 12.43 32.64 40.31 Uterus 35 of 56 21 of 56 32.20 44.73 143.10WBC  1 of 41 40 of 41 −18.91 −13.33 −6.24

TABLE 3b e-Northern for 35832_at: LBFL304 Gene Expression in NormalTissues Global Present Lower Upper Fragment Freq. Tissue Present Absent25% Median 75% 35832_at 0.5228 Adipose 26 of 34  8 of 34 29.09 59.5189.67 Adrenal Gland  1 of 12 11 of 12 −11.00 −6.08 8.14 Appendix 1 of 32 of 3 43.26 53.50 66.52 Artery 3 of 4 1 of 4 182.70 291.81 428.36Bladder 5 of 7 2 of 7 56.36 62.71 64.68 Bone 3 of 4 1 of 4 19.34 77.40167.06 Breast 65 of 82 17 of 82 33.67 63.19 108.61 Cerebellum 0 of 5 5of 5 −19.26 −14.78 −13.16 Cervix  69 of 102  33 of 102 18.76 57.45 94.99Colon  85 of 146  61 of 146 10.73 35.22 87.91 Cortex Frontal 1 of 7 6 of7 −5.03 8.78 14.71 Lobe Cortex 0 of 3 3 of 3 −16.73 −16.67 −15.85Temporal Lobe Duodenum 19 of 53 34 of 53 6.47 20.39 41.95 Endometrium 15of 21  6 of 21 31.44 93.20 137.68 Esophagus 15 of 27 12 of 27 5.12 27.0352.89 Fallopian Tube 19 of 47 28 of 47 5.38 22.48 54.99 GallBladder 2 of7 5 of 7 8.71 28.94 50.85 Heart 0 of 3 3 of 3 −35.98 −28.25 −6.72Hippocampus 2 of 5 3 of 5 −7.43 −3.64 5.68 Kidney 28 of 89 61 of 89 1.6720.45 45.18 Larynx 4 of 4 0 of 4 36.13 54.20 79.75 Left Atrium  80 of141  61 of 141 8.32 25.37 52.28 Left Ventricle  0 of 15 15 of 15 −21.85−17.01 −8.17 Liver  2 of 35 33 of 35 −10.51 0.02 8.05 Lung 29 of 93 64of 93 2.56 19.47 43.63 Lymph Node  3 of 12  9 of 12 −17.58 −2.85 9.56Muscles 12 of 42 30 of 42 −13.74 3.99 23.23 Myometrium  92 of 108  16 of108 67.57 129.39 203.58 Omentum 14 of 15  1 of 15 176.65 310.28 368.41Ovary 31 of 74 43 of 74 0.66 27.78 54.33 Pancreas  4 of 33 29 of 33−9.60 2.09 9.94 Placenta 0 of 5 5 of 5 −21.32 −3.06 6.08 Prostate 30 of32  2 of 32 82.37 104.56 190.72 Rectum 37 of 44  7 of 44 51.53 86.73125.67 Right Atrium  69 of 170 101 of 170 −3.30 8.80 33.56 RightVentricle  35 of 160 125 of 160 −11.65 −0.46 16.02 Skin 28 of 61 33 of61 4.22 25.33 67.56 Small Intestine 36 of 67 31 of 67 10.76 33.92 64.75Soft Tissues 4 of 6 2 of 6 25.95 40.91 58.70 Spleen  1 of 29 28 of 29−19.20 −13.77 −6.69 Stomach 16 of 47 31 of 47 −8.30 13.38 47.93 Testis 1of 5 4 of 5 −18.20 5.01 37.66 Thymus  1 of 73 72 of 73 −22.55 −12.50−3.27 Thyroid Gland 14 of 19  5 of 19 45.56 98.30 141.24 Uterus 43 of 5815 of 58 37.47 103.26 180.98 WBC  0 of 43 43 of 43 −33.45 −25.32 −20.23

TABLE 3c e-Northern Data for 48774_at: LBFL305 Gene Expression in NormalTissues Global Present Freq. Tissue Present Absent Lower 25% MedianUpper 75% 0.9444 Adipose 31 of 32  1 of 32 221.82 286.63 380.88 AdrenalGland 12 of 12  0 of 12 162.12 214.21 310.82 Appendix 3 of 3 0 of 3352.94 506.01 633.71 Artery 3 of 3 0 of 3 343.80 419.88 643.55 Bladder 5of 5 0 of 5 221.82 290.82 301.11 Bone 3 of 3 0 of 3 410.63 508.38 662.78Breast 80 of 80  0 of 80 236.84 279.81 338.22 Cerebellum 5 of 5 0 of 5182.09 198.28 283.55 Cervix  97 of 101  4 of 101 179.11 246.50 317.62Colon 146 of 151  5 of 151 247.18 314.49 389.23 Cortex Frontal Lobe 7 of7 0 of 7 222.19 230.28 268.13 Cortex Temporal Lobe 3 of 3 0 of 3 305.66365.62 377.16 Duodenum 58 of 61  3 of 61 206.17 276.14 331.91Endometrium 21 of 21  0 of 21 158.91 193.40 257.17 Esophagus 25 of 27  2of 27 182.29 223.24 303.93 Eallopian Tube 50 of 51  1 of 51 168.69220.72 265.95 GallBladder 7 of 8 1 of 8 237.67 270.08 312.45 Heart 2 of3 1 of 3 44.79 55.84 56.46 Hippocampus 5 of 5 0 of 5 165.94 212.72328.59 Kidney 79 of 86  7 of 86 121.83 158.99 209.67 Larynx 4 of 4 0 of4 140.76 209.46 302.84 Left Atrium 127 of 141  14 of 141 58.48 92.78123.06 Left Ventricle  9 of 15  6 of 15 50.50 74.69 101.06 Liver 27 of34  7 of 34 87.73 146.60 197.27 Lung 92 of 93  1 of 93 365.58 454.87550.48 Lymph Node 11 of 11  0 of 11 493.34 943.95 1141.06 Muscles 19 of39 20 of 39 41.41 64.44 110.74 Myometrium 104 of 106  2 of 106 188.94263.73 322.65 Omentum 15 of 15  0 of 15 198.02 244.56 334.75 Ovary 70 of74  4 of 74 133.44 181.39 246.40 Pancreas 13 of 34 21 of 34 24.30 53.1384.64 Placenta 4 of 5 1 of 5 156.58 174.21 182.71 Prostate 32 of 32  0of 32 184.16 234.60 289.05 Rectum 42 of 43  1 of 43 284.60 365.66 434.01Right Atrium 148 of 169  21 of 169 54.96 89.92 129.13 Right Ventricle132 of 160  28 of 160 55.58 78.85 114.70 Skin 57 of 59  2 of 59 250.81320.57 398.56 Small Intestine 64 of 68  4 of 68 196.50 279.29 393.83Soft Tissues 6 of 6 0 of 6 234.21 307.07 363.66 Spleen 31 of 31  0 of 31775.25 879.84 1022.49 Stomach 41 of 47  6 of 47 137.91 217.53 338.54Testis 5 of 5 0 of 5 326.62 358.69 377.26 Thymus 71 of 71  0 of 71691.12 802.96 984.42 Thyroid Gland 18 of 18  0 of 18 121.11 162.16238.53 Uterus 57 of 58  1 of 58 157.19 202.53 265.91 WBC 38 of 40  2 of40 1863.06 2264.27 2743.82

TABLE 3d e-Northern Data for 48774_at: LBFL306 Gene Expression in NormalTissues Global Lower Upper Present Freq. Tissue Present Absent 25%Median 75% 0.8143 Adipose 31 of 32  1 of 32 184.04 242.67 285.20 AdrenalGland  6 of 12  6 of 12 130.70 157.08 187.26 Appendix 3 of 3 0 of 3259.39 301.05 388.31 Artery 4 of 4 0 of 4 168.95 207.06 295.97 Bladder 7of 8 1 of 8 196.52 239.43 374.54 Bones 4 of 4 0 of 4 209.35 226.22292.55 Breast 60 of 61  1 of 61 238.09 315.29 421.47 Cervix  92 of 102 10 of 102 180.90 224.31 342.25 Colon 168 of 192  24 of 192 147.41175.83 215.95 Cortex Frontal Lobe 5 of 5 0 of 5 133.94 148.22 162.91Cortex Temporal Lobe 3 of 3 0 of 3 137.28 147.77 172.44 Duodenum 68 of68  0 of 68 186.24 218.73 336.00 Endometrium 19 of 19  0 of 19 273.57359.17 436.85 Esophagus 14 of 25 11 of 25 105.83 140.56 195.89 GallBladder 7 of 8 1 of 8 232.71 293.00 410.47 Heart 1 of 3 2 of 3 86.39100.48 129.68 Hippocampus  9 of 10  1 of 10 125.62 140.94 194.06 Kidney53 of 91 38 of 91 108.29 147.35 186.69 Larynx 3 of 4 1 of 4 160.96190.50 219.55 Left Atrium  65 of 143  78 of 143 100.94 128.86 157.57Left Ventricle  0 of 13 13 of 13 82.54 106.63 117.06 Liver 15 of 44 29of 44 145.82 204.77 244.58 Lung 104 of 114  10 of 114 168.03 203.35283.11 Lymph Node 14 of 14  0 of 14 207.66 319.53 366.70 Lymphocytes(B +T Cells) 24 of 24  0 of 24 224.46 292.01 348.07 Muscles 31 of 40  9 of40 183.43 240.15 329.99 Myometrium 122 of 128  6 of 128 209.83 244.58294.20 Omentum 13 of 15  2 of 15 162.83 236.23 265.24 Ovary 80 of 81  1of 81 219.28 259.00 331.64 Pancreas  8 of 40 32 of 40 97.07 136.80174.54 Prostate 47 of 47  0 of 47 318.31 397.81 525.43 Rectum 38 of 46 8 of 46 143.79 188.95 232.42 Right Atrium  87 of 162  75 of 162 104.22132.56 161.54 Skin 38 of 44  6 of 44 162.29 198.90 236.86 SmallIntestine 72 of 79  7 of 79 184.41 230.96 270.34 Soft Tissues 5 of 5 0of 5 240.30 258.33 499.21 Spleen 36 of 36  0 of 36 253.03 322.87 390.61Stomach 32 of 54 22 of 54 139.62 174.76 239.47 Thymus 70 of 70  0 of 70352.12 438.94 511.11 Thyroid Gland 25 of 25  0 of 25 177.32 211.27276.25 Uterus 54 of 56  2 of 56 243.74 312.71 387.50 WBC 21 of 25  4 of25 149.63 176.72 209.49

INDUSTRIAL APPLICAVILITY Example 3

Detection of LBFL301, LBFL304, LBFL305 or LBFL306 mRNA for StomachCancer Screening

The expression level of mRNA corresponding to SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 17, 19 or 21 is determined in stomach tissue biopsy samples, asdescribed in Example 1, i.e., by screening mRNA samples on a GeneChip,or as described in Example 2, i.e., by screening mRNA samples on aNorthern blot. Alternatively, samples from non-stomach hyperplastictissues in malignant or non-malignant states may also be analyzed.Stomach tissue samples from patients with stomach cancer and from normalsubjects may be used as positive and negative controls. Using any meansof analyzing gene expression, a level of expression higher than that ofthe normal control is indicative of stomach cancer or a likelihood ofdeveloping stomach cancer.

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited patents, patent applications and publications referred to in thisapplication are herein incorporated by reference in their entirety.

1. An isolated nucleic acid molecule selected from the group consistingof: (a) an isolated nucleic acid molecule comprising SEQ ID NO: 3, 5, 7,9, 11, 13, 17 or 19; (b) an isolated nucleic acid molecule that encodesthe amino acid sequence of SEQ ID NO: 4, 14 or 18; (c) an isolatednucleic acid molecule that encodes a protein that is expressed instomach cancer and that exhibits at least about 92% nucleotide sequenceidentity over the entire length of SEQ ID NO: 3 or 17; (d) an isolatednucleic acid molecule that encodes a protein that is expressed instomach cancer and that exhibits at least about 95% nucleotide sequenceidentity over the entire length of SEQ ID NO: 13; (e) an isolatednucleic acid molecule comprising the complement of a nucleic acidmolecule of (a), (b), (c) or (d).
 2. The isolated nucleic acid moleculeof claim 1, wherein the nucleic acid molecule comprises nucleotides174-584 of SEQ ID NO:
 3. 3. The isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule consists of nucleotides 174-584 ofSEQ ID NO:
 3. 4. The isolated nucleic acid molecule of claim 1, whereinthe nucleic acid molecule comprises nucleotides 174-587 of SEQ ID NO: 3.5. The isolated nucleic acid molecule of claim 1, wherein the nucleicacid molecule is selected from the group consisting of: a nucleic acidmolecule consisting of nucleotides 38-892 of SEQ ID NO: 5 and a nucleicacid molecule consisting of nucleotides 38-895 of SEQ ID NO:
 5. 6. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule is selected from the group consisting of a nucleic acidmolecule consisting of nucleotides 53-892 of SEQ ID NO: 7 and a nucleicacid molecule consisting of nucleotides 53-895 of SEQ ID NO:
 7. 7. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule is selected from the group consisting of: a nucleic acidmolecule consisting of nucleotides 65-892 of SEQ ID NO: 9 and a nucleicacid molecule consisting of nucleotides 65-895 of SEQ ID NO:
 9. 8. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule is selected from the group consisting of: a nucleic acidmolecule consisting of nucleotides 92-892 of SEQ ID NO: 11 and a nucleicacid molecule consisting of nucleotides 92-895 of SEQ ID NO:
 11. 9. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule comprises nucleotides 49-1434 of SEQ ID NO:
 13. 10. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule consists of nucleotides 49-1437 of SEQ ID NO:
 13. 11. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule comprises nucleotides 49-1437 of SEQ ID NO:
 13. 12. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule comprises nucleotides 75-575 of SEQ ID NO:
 17. 13. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculeconsists of nucleotides 75-575 of SEQ ID NO:
 17. 14. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises nucleotides 75-572 of SEQ ID NO:
 17. 15. The isolated nucleicacid molecule of claim 1, wherein said nucleic acid molecule is operablylinked to one or more expression control elements.
 16. A vectorcomprising an isolated nucleic acid molecule of claim
 1. 17. A host celltransformed to contain the nucleic acid molecule of claim
 1. 18. A hostcell comprising a vector of claim
 16. 19. A host cell of claim 18,wherein said host cell is selected from the group consisting ofprokaryotic host cells and eukaryotic host cells.
 20. A method forproducing a polypeptide or protein comprising culturing a host celltransformed with the nucleic acid molecule of claim 1 under conditionsin which the polypeptide or protein encoded by said nucleic acidmolecule is expressed.
 21. The method of claim 20, wherein said hostcell is selected from the group consisting of prokaryotic host cells andeukaryotic host cells.
 22. An isolated polypeptide or protein producedby the method of claim
 21. 23. An isolated polypeptide or proteinselected from the group consisting of: (a) an isolated polypeptide orprotein comprising the amino acid sequence of SEQ ID NO: 4, 6, 8, 10,12, 14 or 18; (b) an isolated polypeptide or protein exhibiting at leastabout 92% amino acid sequence identity with SEQ ID NO: 4; (c) anisolated polypeptide or protein consisting of amino acids 31-137 of SEQID NO: 4; (d) an isolated polypeptide comprising a fragment of at least10 amino acids of SEQ ID NO: 6, 8, 10 or 12; (e) an isolated polypeptidecomprising conservative amino acid substitutions of SEQ ID NO: 6, 8, 10or 12; (f) an isolated polypeptide comprising naturally occurring aminoacid sequence variants of SEQ ID NO: 6, 8, 10 or 12; (g) an isolatedpolypeptide exhibiting at least about 75% amino acid sequence identitywith SEQ ID NO: 6, 8, 10 or 12; (h) an isolated polypeptide or proteinexhibiting at least about 95% amino acid sequence identity with SEQ IDNO: 14; and (i) an isolated polypeptide or protein exhibiting at leastabout 92% amino acid sequence identity with SEQ ID NO:
 18. 24. Anisolated antibody or antigen-binding antibody fragment that binds to apolypeptide or protein of claim 23 or to an isolated polypeptide orprotein comprising the amino acid sequence of SEQ ID NO:
 2. 25. Anantibody of claim 24 wherein said antibody is a monoclonal or apolyclonal antibody.
 26. A method of identifying an agent whichmodulates the expression of a nucleic acid encoding a protein of claim23, a protein comprising the amino acid sequence of SEQ ID NO: 2, 20 or22, or a Mst1 protein or a Mst1 splice variant protein, the methodcomprising: exposing cells which express the nucleic acid to the agent;and determining whether the agent modulates expression of said nucleicacid, thereby identifying an agent which modulates the expression of anucleic acid encoding the protein.
 27. A method of identifying an agentwhich modulates the level of or at least one activity of a protein ofclaim 23, or of a protein comprising the amino acid sequence of SEQ IDNO: 2, 20 or 22, or of a Mst1 protein or a Mst1 splice variant protein,the method comprising: exposing cells which express the protein to theagent; determining whether the agent modulates the level of or at leastone activity of said protein, thereby identifying an agent whichmodulates the level of or at least one activity of the protein.
 28. Themethod of claim 27, wherein the agent modulates one activity of theprotein.
 29. A method of identifying binding partners for a protein ofclaim 23 or a protein comprising the amino acid sequence of SEQ ID NO:2, the method comprising: exposing said protein to a potential bindingpartner; and determining if the potential binding partner binds to saidprotein, thereby identifying binding partners for the protein.
 30. Amethod of modulating the expression of a nucleic acid encoding a proteinof claim 23, a protein comprising the amino acid sequence of SEQ ID NO:2, 20 or 22, or a Mst1 protein or a Mst1 splice variant protein, themethod comprising: administering an effective amount of an agent whichmodulates the expression of a nucleic acid encoding the protein.
 31. Amethod of modulating at least one activity of a protein of claim 23, orof a protein comprising the amino acid sequence of SEQ ID NO: 2, 20 or22, or of a Mst1 protein or a Mst1 splice variant protein, the methodcomprising: administering an effective amount of an agent whichmodulates at least one activity of the protein.
 32. A non-humantransgenic animal modified to contain a nucleic acid molecule ofclaim
 1. 33. The transgenic animal of claim 32, wherein the nucleic acidmolecule contains a mutation that prevents expression of the encodedprotein.
 34. A method of diagnosing a disease state in a subject,comprising: determining the level of expression of a nucleic acidmolecule of claim 1, or a nucleic acid molecule encoding a Mst1 proteinor a Mst1 splice variant protein, or of a nucleic acid molecule encodingan isolated polypeptide or protein selected from the group consistingof: (a) an isolated polypeptide or protein comprising the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 18; (b) an isolatedpolypeptide or protein exhibiting at least about 92% amino acid sequenceidentity with SEQ ID NO: 4; (c) an isolated polypeptide or proteinconsisting of amino acids 31-137 of SEQ ID NO: 4; (d) an isolatedpolypeptide comprising a fragment of at least 10 amino acids of SEQ IDNO: 6, 8, 10 or 12; (e) an isolated polypeptide comprising conservativeamino acid substitutions of SEQ ID NO: 6, 8, 10 or 12; (f) an isolatedpolypeptide comprising naturally occurring amino acid sequence variantsof SEQ ID NO: 6, 8, 10 or 12; (g) an isolated polypeptide exhibiting atleast about 75% amino acid sequence identity with SEQ ID NO: 6, 8, 10 or12; (h) an isolated polypeptide or protein exhibiting at least about 95%amino acid sequence identity with SEQ ID NO: 14; and (i) an isolatedpolypeptide or protein exhibiting at least about 92% amino acid sequenceidentity with SEQ ID NO: 18, or of a protein comprising the amino acidsequence of SEQ ID NO: 2, 20 or 22 or of a Mst1 protein or a Mst1 splicevariant protein.
 35. The method of claim 34, wherein the disease stateis stomach cancer.
 36. The method of claim 34, wherein the disease stateis advanced gastric cancer.
 37. The method of claim 34, wherein thedisease state is a malignant neoplasm.
 38. The method of claim 37,wherein the malignant neoplasm occurs in soft tissue, bone, breast,cervix, colon, endometrium, esophagus, kidney, larynx, liver, lung,omentum, ovary, pancreas, rectum, thyroid, myometrium, prostate, skin,small intestine, bladder, spleen or stomach.
 39. The method of claim 20,wherein the splice variant is SEQ ID NO: 13 or SEQ ID NO:
 14. 40. Acomposition comprising a diluent and a polypeptide or protein selectedfrom the group consisting of: (a) an isolated polypeptide or proteincomprising the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14,18, 20 or 22 (b) an isolated polypeptide or protein exhibiting at leastabout 92% amino acid sequence identity with SEQ ID NO: 4 or 18, (c) anisolated polypeptide or protein consisting of amino acids 31-137 of SEQID NO: 4, (d) an isolated polypeptide comprising a fragment of at least10 amino acids of SEQ ID NO: 6, 8, 10 or 12, (e) an isolated polypeptidecomprising conservative amino acid substitutions of SEQ ID NO: 6, 8, 10or 12, (f) an isolated polypeptide comprising naturally occurring aminoacid sequence variants of SEQ ID NO: 6, 8, 10 or 12, (g) an isolatedpolypeptide exhibiting at least about 75% amino acid sequence identitywith SEQ ID NO: 6, 8, 10 or 12, (h) an isolated polypeptide exhibitingat least about 95% amino acid sequence identity with SEQ ID NO: 14.